Ibudilast Reduces IL-6 Levels and Ameliorates Symptoms in Lipopolysaccharide-Induced Sepsis Mice

Abstract

In Japan, ibudilast (IBD) is a therapeutic agent used to treat asthma, allergic conjunctivitis, and dizziness caused by cerebrovascular disease. Previously, we have reported that IBD could reduce the secretion of proinflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in lipopolysaccharide (LPS)-treated RAW264.7 monocyte-linage cells in vitro. In the present study, we examined the anti-inflammatory effects of IBD in vivo. As IL-6 is a biomarker for sepsis and has been suggested to exacerbate symptoms, we determined whether IBD reduces IL-6 levels in vivo and improves sepsis symptoms in animal models. We observed that IBD treatment reduced IL-6 levels in the lungs of LPS-treated mice and improved LPS-induced hypothermia, one of the symptoms of sepsis. In addition, IBD reduced IL-6 and attenuated plasminogen activator inhibitor-1 (PAI-1) and alanine aminotransferase (ALT) levels in the serum of LPS-treated mice. Elevated PAI-1 levels exacerbate sepsis-induced disseminated intravascular coagulation (DIC), and ALT is a biomarker for liver dysfunction. IBD improved the survival of mice administered a lethal dose of LPS. IBD administration ameliorated kidney pathology of model mice. Overall, these results suggest that IBD exerts anti-inflammatory functions in vivo and could be a drug candidate for treating endotoxemia, including sepsis.

INTRODUCTION

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.¹⁾ Despite the use of antibiotics, glucocorticosteroids, and supportive care for treating sepsis, the high mortality rate reportedly persists.^2,3) Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria, the predominant sepsis-inducing microorganism. LPS often causes an uncontrolled inflammatory response, organ failure, and death.⁴⁾ Accordingly, drugs that can potentially regulate the overwhelming inflammatory response in patients with sepsis are urgently needed; however, no specific drugs have been developed.⁵⁾

Ibudilast (IBD) is used to treat asthma, allergic conjunctivitis, and dizziness caused by cerebrovascular disease in Japan. In addition, this drug was recently considered for several inflammatory diseases, including multiple sclerosis⁶⁾ and neuropathic pain.⁷⁾ In a previous study, we have reported that IBD could reduce the secretion of proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α in LPS-treated RAW264.7 cells.⁸⁾ Among proinflammatory cytokines, IL-6 is the most reliable biomarker for sepsis.^9,10) Furthermore, it has been suggested that IL-6 could exacerbate endotoxemia in a model animal.¹¹⁾ Herein, we determined whether IBD exerts anti-inflammatory functions in vivo using an LPS-induced sepsis mouse model^12–15) and assessed its potential as a drug candidate for treating sepsis.

MATERIALS AND METHODS

Reagents

IBD was purchased from Tokyo Chemical Industry (Tokyo, Japan). LPS was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.).

Animal Experiments

Male C57BL/6J mice were purchased from SLC (Shizuoka, Japan) at 6–7-weeks of age, maintained under controlled temperature and light (23 °C and 12-h light–dark, respectively), and used at 7–8-weeks of age; all animals received food and water ad libitum. All experimental procedures followed the guidelines for animal experimentation of the Animal Care and Use Committee of Matsuyama University (Matsuyama, Ehime, Japan). The certification numbers verifying study approval are as follows: 22-007 in 2022.2.22., 21-009 on 2021.3.26., 20-008 on 2020.3.24., and 18-012 on 2019.3.22.

Mice were intraperitoneally (i.p.) administered IBD (0, 20, 100, or 1000 µg/kg) in 0.5% (w/v) methylcellulose 400 solution, sterilized (WAKO 133-17815, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). LPS (0, 5, or 12 mg/kg) in phosphate-buffered saline (PBS) was administered i.p. after 4 h. Twenty-four hours later, the rectal temperature was monitored using a thermometer (BAT-7001H THERMOMETER, PHYSITEMP INSTRUMENTS INC, Clifton, NJ, U.S.A.). Subsequently, a whole blood sample was collected from the heart for serum preparation, and organs, including the lung, kidney, and liver, were harvested for extract preparation or histological analysis.

Protein Extraction from Lung Tissues for Enzyme-Linked Immunosorbent Assay (ELISA)

In brief, harvested mouse lungs were quickly frozen in liquid nitrogen and stored in a deep freezer. Proteins in lung tissue samples were extracted using a 50-fold volume of radio immunoprecipitation assay (RIPA) buffer without sodium dodecyl sulfate and used for ELISA.

Histology

Mouse kidneys and liver were harvested and immersed in a 4% paraformaldehyde solution in PBS for 2 d. The fixed organs were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E).

ELISA

Plasminogen activator inhibitor-1 (PAI-1), TNF-α, IL-10, and IL-6 in the serum or lung extracts were measured using a mouse serpin E1/PAI-1 DuoSet ELISA, 5 Plate (R&D systems #DY3828-05, Bio-techne, Minneapolis, MN, U.S.A.), Mouse TNF-α ELISA MAX Deluxe Set (Bio Legend #430904), Mouse IL-10 ELISA MAX Deluxe Set (Bio Legend #431414), and Mouse IL-6 ELISA MAX Deluxe Set (Bio Legend #431304), respectively.

Measurement of Alanine Transaminase (ALT)

Serum ALT levels were measured using Transaminase CII-Test WAKO (#431-30901) (FUJIFILM Wako Pure Chemical Corporation).

Statistical Analyses

Data are expressed as the mean ± standard error of the mean (S.E.M.). Data values were analyzed using one-factor ANOVA, followed by Tukey’s multiple comparison test. Data from survival experiments were presented using the Kaplan–Meier survival curve, and statistical significance was determined using the log-rank test. p < 0.05 was considered significant.

RESULTS

IBD reduces proinflammatory cytokine levels and ameliorates symptoms in an LPS-induced sepsis mouse model.

Previously, we have reported that IBD reduces the secretion of proinflammatory cytokines such as IL-6 and TNF-α in LPS-treated RAW264.7 monocyte-linage cells in vitro. Accordingly, we investigated the anti-inflammatory functions of IBD in vivo. To assess the anti-inflammatory function of IBD, we used an LPS (5 mg/kg)-induced sepsis mouse model.

As reported by other groups,^11,12) administration of LPS reduces rectal temperature. We observed that IBD ameliorated this reduction in temperature (Fig. 1A). Administration of LPS upregulated IL-6 levels, whereas IBD reduced the upregulated IL-6 levels (Fig. 1B). In addition, LPS minimally induced TNF-α; IBD did not significantly affect TNF-α levels (Fig. 1C). Furthermore, IBD did not affect the levels of IL-10, an anti-inflammatory cytokine^16,17) (Fig. 1D).

Fig. 1. Effect of IBD on Hypothermia in LPS-Challenged Mice and IL-6, TNF-α, and IL-10 Levels in the Lung Tissue from LPS-Challenged Mice

Mice were administered IBD (100 or 1000 µg/kg) 4 h before the LPS challenge (5 mg/kg). (A) Rectal temperature was monitored 24 h after the LPS challenge. (B–D) The levels of IL-6, TNF-α, and IL-10 in the lung tissue of LPS-challenged mice were measured by ELISA 24 h after the LPS challenge. Data are presented as mean ± standard error of the mean (S.E.M.) (n = 5). Asterisks indicate statistically significant differences (* p < 0.05). IBD, ibudilast; IL-6, interleukin-6; IL-10, interleukin-10; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α.

Next, we examined serum levels of sepsis-related biomarkers, including IL-6, PAI-1, and ALT. Administration of LPS upregulated serum IL-6 levels, while IBD treatment reduced these upregulated IL-6 levels (Fig. 2A). Serum levels of PAI-1, a coagulation cascade activator, showed a similar trend to serum IL-6 levels, although no significant differences were observed between the LPS- and the LPS plus IBD-treated groups (Fig. 2B). Serum levels of ALT, a biomarker for liver damage, were also upregulated following LPS administration, and IBD treatment tended to reduce the upregulated ALT levels; however, no significant differences were observed between the LPS- and LPS plus IBD-treated groups (Fig. 2C).

Fig. 2. Effect of IBD on IL-6, PAI-1, and ALT Levels in the Serum of LPS-Challenged Mice

Mice were administered IBD (20 or 100 µg/kg) 4 h before the LPS challenge (5 mg/kg). The levels of IL-6 (A), PAI-1 (B), and ALT (C) in the serum of mice were measured 24 h after the LPS challenge. Data are presented as mean ± standard error of the mean (S.E.M.) (n = 6). Asterisks indicate statistically significant differences (* p < 0.05). ALT, alanine aminotransferase; IBD, ibudilast; IL-interleukin-6; LPS, lipopolysaccharide; PAI-1, plasminogen activator inhibitor-1; TNF-α, tumor necrosis factor-α.

IBD improves the survival rate and kidney pathology of LPS-induced sepsis mice

Based on these findings, we further investigated whether IBD could increase the survival rate of mice with lethal sepsis administered high-dose LPS (12 mg/kg). Forty-eight hours after the LPS challenge, LPS-treated mice showed a survival rate of 43%. Administration of IBD (100 µg/kg) increased the survival rate of LPS-treated mice to 83% at 48 h after the LPS challenge. Administration of a lower dose of IBD (20 µg/kg) also increased the survival rate, although no significant differences were observed between the LPS- and LPS plus IBD (20 mg/kg)-treated groups (Fig. 3A). LPS challenge did not induce clear inflammatory cell infiltration in the kidney or liver under experimental conditions. H&E sections from the kidney of LPS-treated mice showed abnormal glomerular morphology and edema in an extensive area of the organ. Kidney pathology was completely rescued following treatment with 100 µg/kg IBD (Fig. 3B). LPS also induced morphological changes in the liver; however, we failed to detect a notable improvement in liver H&E sections.

Fig. 3. IBD Enhances the Survival Rate of Mice Challenged with a Lethal Dose of LPS and Improves Histological Abnormalities in the Kidney of the LPS-Challenged Mice

Mice were administered IBD (20 or 100 µg/kg) 4 h before the LPS challenge (12 mg/kg). (A) Kaplan–Meier survival curves were used to compare the LPS- and LPS plus IBD-treated groups (n = 12 each). Asterisks indicate statistically significant differences between the LPS- and LPS plus IBD-treated groups (100 µg/kg) (*: p < 0.05). (B) Mouse kidneys and liver were harvested 24 h after the LPS challenge and processed for histological examination. Note that LPS-induced morphological changes in the glomerulus (arrows) and congestion (arrowheads) in the kidney (b) are recovered following treatment with IBD (100 µg/kg) (c), but no notable changes can be seen in terms of LPS-induced liver morphology (e, f). IL-interleukin-6; LPS, lipopolysaccharide.

DISCUSSION

In a previous study, we demonstrated that LPS induces the secretion of IL-6 and TNF-α in RAW264.7 monocyte-linage cells in vitro. In an in vivo pilot study, we confirmed the time-dependent change of IL-6 and TNF-α levels in serum as previously reported^18,19) (data not shown). Serum IL-6 and TNF-α levels peaked 1 to 2 h after LPS challenge and decreased thereafter. TNF-α levels were lower than IL-6 levels, approximately a few hundredth of that of IL-6. In the present study, we detected IL-6 in serum 24 h after LPS challenge (Fig. 2A), confirming the IL-6-inducing effect of LPS in vivo. However, we failed to detect TNF-α in serum at the same time point after LPS challenge. This discrepancy may be attributed to the difference in target cells between in vitro and in vivo experiments. Monocyte-linage cells were the only target of LPS in vitro, while various types of cells were the targets of LPS in vivo.

Hypothermia, a symptom of sepsis,¹⁾ has been documented in LPS-induced mouse models.¹²⁾ We observed that IBD enhanced recovery of LPS-induced hypothermia. Honda et al. have reported that marginal zone B cells in the spleen produce IL-6 in response to LPS and exacerbate endotoxic shock, including hypothermia.¹¹⁾ In a preliminary experiment, we confirmed that IBD reduced the IL-6 level in the spleen of LPS-treated mice (data not shown), suggesting that IBD ameliorates symptoms by reducing IL-6 expression in marginal zone B cells in the spleen; however, further experiments are needed before definite conclusions can be established.

Cytokine release syndrome (CRS) is a life-threatening complication induced by a systemic inflammatory response to infections. The pathogenesis of CRS in patients with sepsis involves IL-6-mediated production of PAI-1 and hyperinflammatory cytokines.¹⁰⁾ PAI-1 is a coagulation cascade activator and a prognostic marker in septic shock.²⁰⁾ Elevated PAI-1 levels exacerbate the progression of systemic inflammation, especially sepsis-induced disseminated intravascular coagulation (DIC).¹⁰⁾ The fibrinolysis-suppressive DIC in the microvasculature, i.e., septic coagulopathy, is often complicated by multiple organ failure (MOF),²¹⁾ which is one of the most important features of sepsis.¹⁾ Thus, IBD-induced reduction of PAI-1 levels in LPS-treated mice could be mediated via decreased IL-6 levels (Figs. 2A, B), potentially improving MOF, including the liver and kidney morphologies as evidenced by ALT levels and improved pathology, respectively (Figs. 2B, 3B).

We detected a discrepancy in the effective dose of IBD with respect to IL-6, PAI-1, and ALT levels, as well as survival rates. IBD (100 µg/kg) increased the survival rate of LPS-challenged mice; however, low-dose IBD (20 µg/kg) was less effective (Fig. 3A). In contrast, low-dose IBD (20 µg/kg) reduced IL-6, PAI-1, and ALT levels in treated mice. High-dose IBD (100 µg/kg) was found to be less effective (Fig. 2). The reason underlying the decreased effectiveness of IBD at the higher dose is not clear. However, we speculate it may be due to adverse drug reactions. From an evaluation standpoint, the observed differences in optimal doses could be explained as follows: low-dose IBD would be desirable for inducing IL-6-, PAI-1-, and ALT-reducing effects. However, in the survival experiment, the low dose of IBD might be degraded before the induction of LPS effects. On administering high-dose IBD, the drug was available after the induction of LPS effects. The development of a more effective IBD regimen is needed to increase the survival rate of patients with sepsis.

Several mechanisms may be involved in the sepsis healing action of IBD. First, IBD is known to be a non-selective phosphodiesterase (PDE) inhibitor.²²⁾ Other PDE inhibitors, such as lisofylline and pentoxifylline, have been reported to ameliorate symptoms in animal models of sepsis.^23,24) Thus, PDE could be a candidate target for the sepsis healing action of IBD. Second, the importance of macrophage migration inhibitory factor (MIF) action has been reported in an in vivo mouse model of severe sepsis.^19,25) IBD has been shown to allosterically inhibit MIF by X-ray crystallography,²⁶⁾ suggesting that MIF could be another candidate target for the sepsis healing action of IBD. Finally, IBD suppresses doxorubicin-induced reactive oxygen species (ROS) production and cytotoxicity by suppressing the formation of transient receptor potential C3 channel and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 protein complexes.²⁷⁾ Furthermore, it has been reported to prevent the delayed increase in baseline minimum alveolar concentration produced by remifentanil,²⁸⁾ suppress mucin production through inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation,²⁹⁾ and attenuate folic acid-induced acute kidney injury through toll-like receptor 4-mediated nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways.³⁰⁾ Therefore, these functions of IBD might be involved in its sepsis healing effect.

In Japan, IBD is used to treat asthma, allergic conjunctivitis, and dizziness. Herein, we demonstrated that IBD has anti-inflammatory functions in vivo and could be a potential drug candidate for treating endotoxemia, including sepsis.

Acknowledgments

This study was supported by JSPS KAKENHI (Grant No. JP19K11707).

Conflict of Interest

The authors declare no conflict of interest.

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