Suppressive Effect of Bortezomib on LPS-Induced Inflammatory Responses in Horses

Accepted Manuscript Suppressive effect of bortezomib on LPS-induced inflammatory responses in horses Hiroaki Sato, Kenshiro Matsuda, Yosuke Amagai, Akane Tanaka, Hiroshi Matsuda

PII:

S0737-0806(16)30696-7

DOI:

10.1016/j.jevs.2017.05.003

Reference:

YJEVS 2321

To appear in:

Journal of Equine Veterinary Science

Received Date: 13 December 2016 Revised Date:

9 May 2017

Accepted Date: 10 May 2017

Please cite this article as: Sato H, Matsuda K, Amagai Y, Tanaka A, Matsuda H, Suppressive effect of bortezomib on LPS-induced inflammatory responses in horses, Journal of Equine Veterinary Science (2017), doi: 10.1016/j.jevs.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Suppressive effect of bortezomib on LPS-induced inflammatory responses in

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horses

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Hiroaki Satoa, Kenshiro Matsudaa, Yosuke Amagaib, Akane Tanakaa,c, Hiroshi

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Matsudaa,b,*

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a

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Bio-Applications and System Engineering, Tokyo University of Agriculture and

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Technology, Fuchu, Tokyo, Japan

Advanced

Health Science,

Graduate School

of

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Cooperative Major

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b

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Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and

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Technology, Fuchu, Tokyo, Japan

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c

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Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu,

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Tokyo, Japan

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Laboratory of Comparative Animal Medicine, Division of Animal Life Science,

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Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of

* Corresponding author at: Hiroshi Matsuda, Laboratory of Veterinary Molecular

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Pathology and Therapeutics, Division of Animal Life Science, Tokyo University of

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Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.

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E-mail address: [email protected] (H. Matsuda).

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Abstract

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Equine endotoxemia is a serious clinical problem with high mortality. Only a few

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treatments have been proved the therapeutic efficacy for equine endotoxemia.

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Excessive nuclear factor kappa B (NF-κB) activation and production of

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proinflammatory cytokines, which were induced by reaction to lipopolysaccharide

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(LPS), would play a key role in the pathogenesis of endotoxemia. Bortezomib, a

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proteasome inhibitor, inhibits NF-κB signaling pathway through blocking

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proteasomal degradation of NF-κB inhibitor alpha. This study aimed to evaluate the

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effect of bortezomib on TNF-α production by LPS-stimulated equine monocytes in

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vitro and on clinical and inflammatory parameters in an in vivo endotoxemia model.

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Bortezomib significantly inhibited LPS-induced TNF-α production through

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inhibition of NF-κB activity in vitro. In a cross over design, horses received

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pretreatment of either bortezomib (1.3 mg/m2) or vehicle (dimethyl sulfoxide) prior

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to the infusion of 30 ng/kg LPS. Clinical parameters including behavioral pain

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scores and hoof wall surface temperature (HWST) were measured over 7 h. In an

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endotoxemia model, bortezomib had a tendency to improve painful reaction and

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reduction of HWST. Bortezomib would have a potential as a therapeutic agent for

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equine endotoxemia.

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Horse, Endotoxemia, Proteasome inhibitor, Tumor necrosis factor-alpha, Nuclear

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factor-kappa B

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1. Introduction Equine endotoxemia is one of the intractable diseases with still high mortality [1,

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2]. Severe colic frequently leads equine endotoxemia that presents complex clinical

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signs [1]. Lipopolysaccharide (LPS), a component of the outer cell membrane of

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Gram-negative bacteria such as Escherichia coli, is a major trigger of endotoxemia.

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Overproduction of proinflammatory cytokines produced by immunocompetent cells

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in response to LPS cause systemic inflammatory response syndrome with

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cardiovascular depression, arterial hypoxemia, decreased tissue perfusion, and

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peripheral hypoxia; in the worst case, resulting in multiple organ dysfunction and

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death [1]. It is well known that LPS induces nuclear factor kappa B (NF-κB)

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activation in monocytes/macrophages leading to production of proinflammatory

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cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6.

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In particular, TNF-α strongly correlates with clinical signs of equine endotoxemia

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[3]. Hence, excessive activation of NF-κB would play a key role in the pathogenesis

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of LPS-mediated diseases such as endotoxemia.

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The current main treatments for equine endotoxemia are intravenous

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administration of flunixin meglumine (FM) and fluid therapy [4]. However, FM has

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considerable adverse-effects on small intestine functions, such as the increased

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mucosal permeability and the influx of LPS, and thus may paradoxically exacerbate

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existing endotoxemia [5, 6]. Although many therapies have been attempted for

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equine endotoxemia so far, only a few have proven efficacy [2, 4].

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Bortezomib, a first-in-class proteasome inhibitor, was developed as a therapeutic

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agent of multiple myeloma [7, 8]. Molecularly targeted agent bortezomib induces

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apoptosis of multiple myeloma cells through inhibition of degradation of NF-κB

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inhibitor alpha (IκBα) and blocking of NF-κB DNA-binding activity [9-11]. Because

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bortezomib inhibits NF-κB activation, the application for treatment of not only

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cancer but also NF-κB-associated inflammatory disorders is attempted [12, 13].

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Recently it has been demonstrated that local infusion of the other NF-κB inhibitor

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improved LPS-induced digital hypothermia in horses [14], suggesting that NF-κB is

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a potential therapeutic target for treatment of LPS-mediated diseases. The aims of

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this study were to evaluate the effect of bortezomib on LPS-induced TNF-α

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production from equine monocytes in vitro and on clinicopathological parameters in

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an in vivo model of equine endotoxemia. We hypothesized that systemic treatment

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with bortezomib improves pathological conditions in an equine endotoxemia model.

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2. Materials and Methods

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2.1. Animals The study was approved by the University Animal Care and Use Committee of

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the Tokyo University of Agriculture and Technology and was performed in

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accordance with the guidelines and regulation. All horses used in this study were

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diagnosed as healthy ones by physical examinations, gait evaluation, complete blood

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counts, and serum biochemistry profiles.

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2.2. In vitro study

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2.2.1. TNF-α assay

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Blood from four adult healthy gelding horses, 2 thoroughbreds and 2

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warmbloods, was used in in vitro experiments. Mononuclear cells were isolated from

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blood

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diatrizoate solution (Histopaque-1077; Sigma-Aldrich, St. Louis, MO) as previously

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described [15]. Mononuclear cells were suspended in RPMI-1640 (Gibco, Grand

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Island, NY) at a final concentration 4 × 106 cells/ml, and 2 × 106 cells (0.5 ml) were

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placed in each well of 24-well polystyrene plates and incubated for 2 h at 37°C in a

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5% CO2 atmosphere to allow monocyte adherence. Non-adherent cells were removed

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by washing the plate five times with warm RPMI-1640. Adherent monocytes were

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centrifugation

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polysucrose/sodium

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overlaid with RPMI-1640 containing 10% equine serum (HyClone Donor Equine

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Serum; Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 100

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units/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich). Cells are incubated

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in the medium with a range of concentrations of bortezomib (1, 10, 100, and 1,000

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nM; StressMarq Biosciences Inc., British Columbia, Canada) or 0.01% dimethyl

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sulfoxide (DMSO) as a vehicle control in the presence of 1 ng/ml LPS (Escherichia

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coli O55:B5; Sigma-Aldrich). After incubation for 6 h at 37°C in a 5% CO2

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atmosphere, the plate was centrifuged (1,000 rpm, 10 min) and supernatants were

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collected form each well for measurement of TNF-α by an enzyme-linked

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immunosorbent assay (ELISA) kit (Thermo Fisher Scientific Inc.). More than 81%

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and 98% of the adherent cells were nonspecific esterase positive and dye exclusion

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test positive, respectively.

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2.2.2. Western blot analysis

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2.2.2.1. IκBα

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Mononuclear cells were isolated from 3 adult healthy thoroughbreds as described

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above. A total of 1 × 107 cells were placed into each well of 6-well polystyrene

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plates and incubated for 2 h. Adherent monocytes were pretreated with various

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concentrations of bortezomib for 1 h. Cells were stimulated with 1 ng/ml LPS for 5

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min, following the pretreatment. Western blot analysis was performed as previously

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described [16, 17].

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2.2.2.2. NF-κB

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Mononuclear cells were isolated from 3 adult healthy thoroughbreds. A total of 5

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× 106 mononuclear cells were incubated with the optimal dose (1,000 nM) of

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bortezomib in 1.5 ml polypropylene micro tube for 1 h, followed by stimulation with

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1 or 10 ng/ml LPS for 6 h. To prevent activation of NF-κB secondary to monocyte

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adherence, the tubes were rotated during the incubation using a rotation/revolution

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mixer (Revolution Mixer RVM-100; AGC Techno Glass, Shizuoka, Japan). Nuclear

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extraction and Western blot analysis were performed as previously described [16,

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2.3. In vivo study

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2.3.1. Experimental design

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Three adult thoroughbreds (horse A, B, and C) were used in in vivo experiments.

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Table 1 shows the more information of the horses. The study was conducted as a

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crossover design in which each horse acted as its own control. In the first

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experimental period, horse A and B received bortezomib, and horse C received

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vehicle. After a washout period of 6 weeks between experiments, the second

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experiment was crossed over.

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Bortezomib was dissolved at 50 mg/ml in DMSO and stored at −20°C. The

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infusion dose of bortezomib was selected 1.3 mg/m2 body surface area (BSA)

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according to dose on human multiple myeloma [18]. BSA was calculated according

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to the following formula [19].

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BSA (m2) = body weight (bwt; g)2/3 × 10.5 × 10−4

1.3 mg/m2 bortezomib or an equivalent volume of vehicle were diluted with 20 ml of

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0.9% saline, and intravenously infused 30 min (−30 min time point, horse A and B)

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or 60 min (−60 min time point, horse C) prior to the challenge of LPS (0 min time

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point).

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2.3.2. Endotoxemia model The experiments were performed in an air-conditioned, concrete-covered stall

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bedded with wood shavings in an animal medical center. To acclimate to

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environment and room temperature, the horses were placed into the stall for at least

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1 h before the experiments, and the room temperature was kept as constant as

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possible (the intraexperimental coefficient of variation < 2.4%). Endotoxemia was induced by the low-dose LPS challenge as previously reported

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[20-23]. A 14 gauge 13.3 cm angiocatheter (Angiocath; Becton Dickinson Infusion

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Therapy Systems Inc., Franklin lakes, NJ) was placed in the left jugular vein for

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infusions and blood sample collection, under local anesthesia (2% lidocaine

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hydrochloride; AstraZeneca K. K., Osaka, Japan). LPS was infused at a dose 1

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ng/kg/min over 30 min (time point 0 to 30 min; total dose 30 ng/kg). The horses

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were fasted until 270 min time point. After assessment at the time point, they were

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allowed ad libitum access to hay and water. At the 8 h (420 min time point), the

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experiment was finished, and immediately thereafter all horses received intravenous

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injection of 1.1 mg/kg bwt FM (Banamine; Sumitomo Dainippon Pharma Co., Ltd.,

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Osaka, Japan). On the next day of the experiment, the horses underwent physical

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examinations and no abnormalities were detected.

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2.3.3. Clinical parameters

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Clinical parameters including behavioral pain score and hoof wall surface

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temperature (HWST) were recorded at −60, −30, 0 (immediately before LPS

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infusion), 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, and 420 min.

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Behavioral pain scores were assessed using a numerical rating scale with range from

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9 (representing no pain) to 35 (extreme pain), which was previously validated in the

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horse [24]. HWST was measured using a thermographic camera (FLIR i7; FLIR

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Systems Inc., Tokyo, Japan) set 1 m away from the hoof wall and focused on the

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hoof wall surface at one third of the way from the coronary band to the toe of the left

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forelimb, as previously reported [14, 21]. Heart rate, respiratory frequency, and

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rectal temperature were also assessed.

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2.4. Data analysis

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The in vitro data were compared by Williams multiple comparison test. The area

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under the curve values from -30 min to 420 min of clinical parameters were

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compared by Student’s t-test (alpha error, 0.05; beta error, 0.20). Values of P < 0.05

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were considered to be significant.

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3. Results

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3.1. Suppressive effect of bortezomib on TNF-α release from monocytes

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When monocytes were incubated with the optimal dose of LPS for 6 h, TNF-α

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levels in the supernatants were detected by an ELISA.

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significant release of TNF-α, whereas a small amount of TNF-α was detected in the

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control without LPS ranging from 414 to 767 pg/ml. To evaluate efficacy of

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bortezomib, various concentrations of bortezomib were added into the culture and

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relative effectiveness was examined (Fig. 1). Bortezomib inhibited the LPS-induced

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TNF-α production in a dose-dependent manner; and TNF-α levels in the supernatants

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of incubation with 1,000 nM bortezomib were roughly comparable to those without

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LPS.

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not shown).

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There was no significant difference in cell viability between the groups (data

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Addition of LPS induced

3.2. Inhibition of IκBα and NF-κB activation by bortezomib To examine the action of bortezomib, IκBα phosphorylation and NF-κB nuclear

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translocation were detected by western blot analysis (Fig. 2). Addition of bortezomib

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inhibited proteasomal degradation of phosphorylated IκBα of monocytes in a dose

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dependent manner. Bortezomib also inhibited nuclear translocation of NF-κB in

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LPS-stimulated mononuclear cells.

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3.3. Clinical parameters improved by pretreatment with bortezomib

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Horses treated with the optimal dose of LPS manifested typical clinical

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symptoms of endotoxemia [20-23]. After LPS challenge to the control horses

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pretreated with vehicle alone, the pain scores of all the horses reached a maximum at

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90 min: 32 scores in horse A, 29 scores in horse B, and 30 scores in horse C, and

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then decreased (Fig. 3). After allowed ad libitum access to hay and water at 270 min

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after LPS treatment, the scores were slightly increased again. On the other hand,

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horses pretreated with 1.3 mg/m2 BSA bortezomib manifested significantly lower

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pain scores (115.4 ± 4.6 AUC score X h) than those pretreated with vehicle alone

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(142.5 ± 3.9 AUC score X h); the pain scores were reduced 4–8 scores in 90 min

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after the injection with LPS.

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As shown in Fig. 4, HWST in horses pretreated with vehicle decreased in 60 min

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after injection with LPS, reaching a nadir between 150 and 210 min. Conversely,

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horses pretreated with bortezomib showed significantly smaller change in levels of

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HWST (from 2.9 to 4.6°C) (59.9 ± 5.2 AUC °C X h) as compared to those pretreated

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with vehicle alone (42.2 ± 2.0 AUC °C X h).

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There were no significant differences in heart rate, respiratory frequency, and

rectal temperature between the two groups (data not shown).

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4. Discussion The experimental equine endotoxemia model used in this study has previously

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been developed by many investigators [3, 20-23]. This model was characterized by

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high reproducibility of clinical signs (pyrexia, tachycardia, painful reaction, and

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digital hypoperfusion) and inflammatory reaction (leukocyte activation and increase

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in proinflammatory cytokine production), and therefore it is selectively used to

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assess the therapeutic efficacy of candidate agents for equine endotoxemia. The

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findings in this study including pyrexia and tachycardia (data not shown) were

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similar to those reported previously.

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Proinflammatory cytokine TNF-α plays a crucial role in the pathogenesis of

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equine endotoxemia. It is well known that LPS activates NF-κB signaling in immune

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cells through toll-like receptor 4 of the surface [25]. Under the unstimulated

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condition, NF-κB exists as an inactive form in the cytoplasm by binding its

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endogenous inhibitor IκB. Following stimulation by LPS, IκB is phosphorylated by

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IκB kinases [26]. Phosphorylated IκB immediately undergoes proteolysis by the 26S

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proteasome and subsequently translocates to the nucleus, where NF-κB induces

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transcription of proinflammatory cytokines and mediators [25, 27]. Therefore,

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prevention of excessive inflammatory responses through blockade of NF-κB

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activation is expected to be beneficial for the treatment of equine endotoxemia. NF-κB inhibition by local treatment with IMD-0354, a synthetic IκB kinase β

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inhibitor [28], is effective to digital vasoconstriction by LPS in horses [14]. Digital

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hypothermia with vasoconstriction was markedly suppressed by the treatment with

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IMD-0354. Therefore, in this study we focused on an agent clinically and

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commercially available. Bortezomib has an inhibitory effect on the activation of

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NF-κB by proteasome blockade. Although this agent was developed as a therapeutic

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candidate for cancers [7, 8], the efficacy on an anterior uveitis model and a sepsis

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model in rodents have been also reported [12, 13].

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this study. The infusion dose of bortezomib is determined with reference to the dose

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on human multiple myeloma [18].

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phase I and II trials showed initial plasma levels of approximately 480 nM following

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administration at a dose of 1.3 mg/m2 [18], which are within the concentration

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range where bortezomib showed inhibition in vitro experiments. The dose of 1.3

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mg/m2 was chosen according to these findings, even though there are species

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differences in drug-metabolizing systems present between humans and horses. The

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pharmacokinetic analysis of bortezomib shows a biphasic elimination profile,

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characterized by a rapid initial distribution phase followed by a longer elimination

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phase. Because the optimal timing of bortezomib administration for equine

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endotoxemia is unknown, the different timings (30 or 60 min prior to LPS challenge)

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of the administration were tested. The administration of bortezomib in horses has not

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been previously reported; therefore, the administration experiment had to be

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carefully conducted. We carried out as a pilot study with a small sample size to

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reduce the risk of unexpected adverse events in horses treated with bortezomib. As

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expected, no severe adverse event was observed during and after this study.

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Bortezomib improved pain score in this study. Pain associated with endotoxemia

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may be caused by prostaglandins [29]. NF-κB activated by LPS or TNF-α induced

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expression of cyclo-oxygenase-2, resulting in prostaglandin synthesis. The mild

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pain-reducing effect of bortezomib might due to inhibiting cyclo-oxygenase-2 via

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blocking NF-κB. The effect of bortezomib was remarkable on decrease in HWST as

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an indirect indicator of digital perfusion [21]. Endothelin-1 is a potent

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vasoconstrictor peptide and an important marker of vascular damage. Increased

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plasma endothelin-1 concentration has been reported in endotoxemic horses [30, 31].

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Because NF-κB mediates endothelin-1 production by vascular endothelial cells [32,

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33], bortezomib may suppress endothelin-1 production via blocking NF-κB

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activation, leading to improvement of digital hypoperfusion. One of the most serious

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complications of endotoxemia is laminitis. Endotoxemia was reported to be an

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important risk factor for the development of acute laminitis in a retrospective study

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[34]. Therefore, bortezomib may prevent the development of laminitis secondary to

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endotoxemia by inhibition of digital hypoperfusion via NF-κB blockade.

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This study will serve as a basis to continue the research evaluating the efficacy.

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Further study with a larger sample size on the pharmacokinetics property, safety,

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effective dose, and administration timing of bortezomib in horses are needed.

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5. Conclusions

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The present study demonstrated that bortezomib inhibited LPS-induced TNF-α

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production from equine monocytes through inhibition of IκBα degradation in vitro,

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and improved painful reaction and reduction of HWST in horses treated with LPS.

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These in vitro and in vivo findings suggest that bortezomib might have a potential as

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a therapeutic agent for equine endotoxemia or endotoxin-associated laminitis.

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Acknowledgments This study was supported by grants from the Grant-in-Aid for Scientific

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Research (#15K14868 and #16H06383) provided by the Japan Society for the

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Promotion of Science. The authors thank Drs. K. Oida, H. Jang and S. Ishizaka for

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their technical advice and support and all members of the equestrian team at the

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Tokyo University of Agriculture and Technology for the care of the horses. No

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financial or personal relationships inappropriately influence or bias the content of

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this research.

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flunixin meglumine, and their combination on a model of endotoxemia in horses.

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Ethyl pyruvate diminishes the inflammatory response to lipopolysaccharide

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Table 1. Horses participated in in vivo experiments Horse

Breed

Age

Sex

Body weight

A

thoroughbred

8

male

500

B

thoroughbred

5

gelding

538

C

thoroughbred

3

female

438

AC C

EP

TE D

M AN U

SC

403

RI PT

402

25

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404

Figure legends

405

Fig. 1. Effect of bortezomib on LPS-induced TNF-α production by equine peripheral

407

blood monocytes. Monocytes were incubated with bortezomib or vehicle in the

408

presence or absence of 1 ng/ml LPS for 6 h. TNF-α levels in supernatants were

409

shown as the relative value to the control (white left column). Each column

410

represents the mean ± SEM of 4 different experiments. *P < 0.05 versus LPS alone.

411

SEM, standard error of the mean.

412

M AN U

SC

RI PT

406

Fig. 2. NF-κB activation. (A) IκBα degradation activity suppressed by bortezomib.

414

After preincubation with the indicated concentrations of bortezomib for 1 h,

415

monocytes were stimulated by 1 ng/ml LPS for 5 min; whole cell lysates were

416

obtained to detect the IκB phosphorylation by Western blot analysis. (B) NF-κB

417

nuclear translocation blocked by bortezomib. After preincubation with 1,000 nM

418

bortezomib for 1 h, mononuclear cells were stimulated by 1 or 10 ng/ml LPS for 6 h;

419

nuclear extracts were obtained to detect the NF-κB activity by Western blot analysis.

420

Results show representative of three independent experiments (three horses).

AC C

EP

TE D

413

421

26

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Fig. 3. Pain scores. Horses were challenged with 30 ng/kg bwt LPS infused over 30

423

min (0–30 min time point) after pretreatment with bortezomib (closed) or vehicle

424

(open). The data for each horse are presented separately. The arrow indicates the

425

timing of pretreatment. Bortezomib generally tended to improve LPS-induced

426

painful reaction.

SC

RI PT

422

427

Fig. 4. HWST. Horses were challenged with 30 ng/kg bwt LPS infused over 30 min

429

(0–30 min time point) after pretreatment with bortezomib (closed) or vehicle (open).

430

The data for each horse are presented separately. The arrow indicates the timing of

431

pretreatment. Bortezomib inhibited LPS-induced decrease of HWST in all 3 horses.

AC C

EP

TE D

M AN U

428

27

SC

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5 4 3

*

2

*

TE D

TNF-α concentration (ng/ml)

6

1

0

AC C

EP

LPS

− + + + + + 0

0

1

10 100 1,000

Bortezomib (nM)

ACCEPTED MANUSCRIPT A

Bortezomib (nM) 0

LPS

0

10 100 1,000

− + + + +

p-IκB

B

Bortezomib

− − + − + LPS

−

1

1

10

10

NF-κB p65

AC C

EP

TE D

M AN U

Lamin B1

SC

β-actin

RI PT

IκB

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Horse A 35

RI PT

30 25 20 15

SC

10 5

Horse B

35 30 25 20 15

TE D

Pain scores

0 60 120 180 240 300 360 420 LPS

M AN U

-60

10 5

-60

0

60 120 180 240 300 360 420

EP

LPS

AC C

35

Horse C

30 25 20 15 10 5 -60

0

60 120 180 240 300 360 420

LPS

Time (min)

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Horse A 4

RI PT

2 0 -2 -4

SC

-6 -8 -60

0

60 120 180 240 300 360 420

M AN U

6

Horse B

4 2 0 -2 -4

TE D

Δ Temperature (°C)

LPS

-6 -8

-60

0

60 120 180 240 300 360 420

EP

LPS

AC C

Horse C

4 2 0

-2 -4 -6 -8 -60

0

60 120 180 240 300 360 420

LPS

Time (min)

ACCEPTED MANUSCRIPT Highlights !

A proteasome inhibitor, bortezomib, inhibited activation of NF-κB.

!

We evaluated the effect of bortezomib on LPS-induced inflammatory responses in horses.

!

Bortezomib inhibited the LPS-induced TNF-α production on equine

!

Bortezomib improved painful reaction and digital hypoperfusion in an endotoxemia model.

!

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monocytes.

Bortezomib

might

have

a

therapeutic

on

endotoxemia

AC C

EP

TE D

M AN U

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LPS-associated laminitis.

effect

1

or