A synergistic effect of Ambroxol and Beta-Glucosylceramide in alleviating immune-mediated hepatitis: A novel immunomodulatory non-immunosuppressive formulation for treatment of immune-mediated disorders

A synergistic effect of Ambroxol and Beta-Glucosylceramide in alleviating immune-mediated hepatitis: A novel immunomodulatory non-immunosuppressive formulation for treatment of immune-mediated disorders:



Publication date: December 2020

Source: Biomedicine & Pharmacotherapy, Volume 132

Author(s): Tawfik Khoury, Yuval Ishay, Devorah Rotnemer-Golinkin, Lidya Zolotarovya, David Arkadir, Ari Zimran, Yaron Ilan




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Biomedicine & Pharmacotherapy
Volume 132, December 2020, 110890

A synergistic effect of Ambroxol and Beta-Glucosylceramide in alleviating immune-mediated hepatitis: A novel immunomodulatory non-immunosuppressive formulation for treatment of immune-mediated disorders
Author links open overlay panelTawfikKhouryaYuvalIshayaDevorahRotnemer-GolinkinaLidyaZolotarovyaaDavidArkadirbAriZimrancYaronIlanaShow more
https://doi.org/10.1016/j.biopha.2020.110890Get rights and content
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open access


Highlights


•

Ambroxol hydrochloride is used in respiratory diseases as a broncholytic therapy.•

Beta-Glucosylceramide (GC) is a glycosphingolipid that exerts an immune protective effect.•

Coadministration of Ambroxol and GC significantly alleviated immune-mediated liver damage.•

A synergistic effect on suppression of intrahepatic CD8+CD25+ was shown.•

Synergism was documented for an increase in the CD4/CD8 lymphocyte ratio.



Abstract

Background

Ambroxol hydrochloride is being used in respiratory diseases as a broncholytic therapy. Beta-Glucosylceramide (GC) is a naturally occurring glycosphingolipid that exerts an immune protective effect. The aim of the present study was to determine the synergistic immunomodulatory effect between these two compounds.

Methods

Immune-mediated hepatitis was induced in the mice by administration of Con A. Mice were treated with either Ambroxol or GC alone or with the combination of both. Mice were followed for their effect on the liver injury, cytokine profile, and the immune system.

Results

Coadministration of Ambroxol and GC significantly alleviated the liver injury induced by ConA, as demonstrated by the decreased liver enzymes. The combined treatment had a statistically significant synergistic effect on the suppression of intrahepatic CD8+CD25+, an increase in the CD4/CD8 lymphocyte ratio and in the CD8+ intrahepatic lymphocyte trapping, as well as on change of serum in the IL4 levels. The beneficial effect was associated with the promotion of regulatory T lymphocytes subsets, and with a trend for a pro-inflammatory to an anti-inflammatory cytokine shift.

Conclusions

Coadministration of Ambroxol with GC exerted a synergistic immunoprotective effect in a model of immune-mediated acute liver damage. Considering the high safety profile of both agents, the combination may become a novel immunomodulatory non-immunosuppressive therapeutic agent.

Significance statement

Coadministration of Ambroxol with glucocerebroside exerted a synergistic immunoprotective effect in a model of immune-mediated acute liver damage.

Graphical abstract



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Abbreviations
GC
Beta- Glucosylceramide
ConA
Concanavalin A
PMNLs
polymorphonuclear leucocytes
IL
interleukin
TNF
tumor necrosis factor
DC
dendritic-cells
NKT
natural killer T cells
IFN
interferon
PBS
phosphate-buffered saline
AST
aspartate aminotransferase
ALT
alanine aminotransferase
FACS
Fluorescence-activated cell sorting
TGF
transforming growth factor

Keywords
Ambroxol
immune mediated hepatitis
glucocerebroside

1. Introduction

The Current methods for the treatment of immune-mediated and autoimmune disorders usually involve the use of immunosuppressive agents. These drugs are associated with side effects, many of which are related to a direct suppressive effect on various arms of the immune system [1]. Therefore, there is a need to develop the immunomodulatory agents, which will not exert immunosuppressive effects.

Ambroxol hydrochloride is being used in several countries as an over-the-counter bronchosecretolytic and expectorant drug [2,3]. In clinical practice, it has been proved to facilitate the repair of bronchial epithelium and to accelerate mucociliary transport [2,3]. Ambroxol stimulates the formation and release of surfactant by type II pneumocytes [4] and is an effective inhibitor of free radical-mediated processes in the lungs [5]. Ambroxol protects α1-proteinase inhibitor from oxidative inactivation and inhibits the chemotactic response and spontaneous migration of human polymorphonuclear leucocytes (PMNLs). It inhibits the interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα) production by mononuclear cells stimulated with lipopolysaccharide (LPS) [6]. Intraperitoneal administration of Ambroxol protects the lung and heart lipids from oxidative stress provoked by intravenous injection of endotoxin in mice [7]. Ambroxol inhibits cigarette smoke-induced acute lung injury in a by inhibiting the Erk pathway, TNF-alpha, and IL-1beta [8]. Ambroxol prevents airway inflammation through alteration of cytokines and protection from oxidative stress. Levels of IL-5 and IL-13 in BAL fluid were decreased, along with increased levels of IL-10 and IL-12 [9].

Beta- glucosylceramide (GC) has been previously shown to exert a beneficial effect on a variety of immune-mediated disorders [[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]]. Preliminary data suggest that it exerts similar effects in humans [22]. GC affects cell membranes and the crosstalk between dendritic cells (DCs) and natural killer T (NKT) cells, which underlie some of its beneficial effects [11,[14], [15], [16], [17],19,23,24].

Concanavalin A (ConA), plant lectin and T cell mitogen, rapidly induces severe immune-mediated hepatitis in the mice that is associated with increased pro-inflammatory cytokines, IFN-γ, IL-12, IL-18, and IL-4 expression [25], and in which NKT lymphocytes, CD4 + T cells, and Kupffer cells have a contributory role [26,27].

The aim of our study was to examine the potential to develop Ambroxol with GC-synergistic combination that will have an immunomodulatory role without immunosuppression.
2. Materials and Methods
2.1. Animals

Male C57Bl/6 mice (12 weeks old) were obtained from Harlan Laboratories (Jerusalem, Israel) and maintained in the Animal Core of the Hadassah-Hebrew University Medical School. The Mice were kept at 12 weeks of age and maintained in the Animal Core of the Hebrew University Medical School. All the mice were administered standard laboratory chow and water ad libitum and kept in a 12 -h light/dark cycle. All the experiments were performed in accordance with the guidelines of the Institutional Committee for Care and Use of Laboratory Animals (IACUC protocol number: MD-16-14986-3).
2.2. Oral administration of Ambroxol hydrochloride

Ambroxol was dissolved in distilled water by vigorous shaking. The Ambroxol solution was orally administered to the mice.
2.3. Preparation of Glycolipids

β-glucosylceramide (GC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). GC was dissolved in a mixture of 30% Cremophor EL (Sigma, Rehovot, Israel) and ethanol (1:1) in PBS.
2.4. Experimental groups

Four groups of mice (n = 4) were studied. Mice in all the groups were injected with Concanavalin A (Con A, 500 mg/mouse). Mice in the control group A were treated with PBS. The Mice in group B were orally administered 6 mg of GC, per mouse daily for 5 days prior to the ConA injection. Mice in group C were orally administered 1.4 mg of Ambroxol per mouse daily for 5 days. Mice in group D were orally administered the combination of 6 mg of GC and 1.4 mg of Ambroxol per mouse daily for 5 days.
2.5. Histological examination of the liver

Livers of all the mice in all the experimental group was cut into 5 μm thin slices, fixed in formaldehyde solution (10%), and kept at room temperature. The Tissue blocks were embedded in paraffin. The sections were stained with hematoxylin-eosin (H&E) for morphological examination.
2.6. Liver enzymes

The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined with an automatic analyzer on all the mice in all groups.
2.7. Assessment of the effect on the systemic immune system

The immune modulatory effect determined by the FACS analysis and serum cytokines.
2.8. Flow cytometry on isolated splenocytes and hepatic lymphocytes

Spleen and liver-derived lymphocytes were isolated as previously described [28,29]. Flow cytometry was performed on lymphocytes resuspended in 1 mL of the FACS buffer (PBS + 1% BSA + 0.1% sodium azide). Cells were stained with the diluted anti-LAP antibody (50 μL /sample), FITC-conjugated anti-CD4/CD8 (0.5 μL per sample), PE-conjugated anti-CD25/NK1.1 FITC–conjugated anti-CD3 (1 μL per sample), and PerCP-conjugated anti-CD45 (2 μL per sample). All stains were performed after blocking the Fc receptor with anti-mouse CD16/CD32 (BD Fc Block). The flow cytometry was performed using an LSR-II flow cytometer and FCS express software.
2.9. Cytokine measurement

Cytokine assessment was performed on serum samples by the MILLIPLEX® Analytes (EMD Millipore Corporation, Missouri 63304 U.S.A.) based on the Luminex xMAP® technology for performing immunoassays on the surface of the fluorescent-coded magnetic beads MagPlex®-C microspheres. The Acquiring and analyzing the data were performed using the Luminex analyzer (MAGPIX®) software. Cytokines assessment was measured by the mean fluorescence intensity (MFI).
2.10. Statistical analysis

SAS Vs 9.14 was used for statistical analysis. A one-way analysis of variance (ANOVA) model was used to analyze differences between groups. For significant ANOVA models, post-hoc comparisons were performed, using the Tukey-Kramer adjustment for multiple comparisons. Statistical analysis was performed by an experienced statistician.
3. Results
3.1. A synergistic effect of oral administration of Ambroxol and GC on


Fig. 1 shows the effects of the treatment on different subsets of lymphocytes. Panel A1 shows a decrease in the hepatic CD8+CD25+ lymphocytes which were noted only in mice treated with the combination of Ambroxol and GC in group D compared to the untreated mice in group A (6.09% vs. 11.25% for groups D and A, respectively). Panel B shows a significant effect on the redistribution of NK1.1 positive cells, both in the spleen and in the liver of the mice in the treated group. There was a decrease in the splenic to the hepatic ratio of NK1. Cells in group B, C and D as compared to group A (2.2% vs. 0.55% vs. 0.13% vs. 0.38%, for groups A, B, C, and D, respectively).
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Fig. 1. Ambroxol and GC alter the systemic and hepatic immune system. FACS analysis was performed on lymphocytes subsets harvested from both the liver and spleen of mice in all groups for CD8+CD25+ (A); Ratio of spleen/liver NK1.1 (B); the ratio of CD4 to CD8 is the spleen and in the liver (C); ratio of splenic CD4/CD8 to hepatic CD4/CD8 ratio (D); and CD3+FoxP3 (E).

Panel C shows a synergistic effect of the combination of Ambroxol and GC on the CD4/CD8 lymphocyte ratio. The CD4\CD8 ratio in the spleen increased in the combination treated group compared to the other groups. No significant effect was noted for the CD4/CD8 ratio in the liver.

Panel D shows the intrasplenic/ intrahepatic ratio of the CD4/CD8 ratios. This ratio was markedly increased only in the combination treated group D compared to the other groups suggesting the sequestration of CD8+ positive cells in the liver.

Panel E shows an increase in the FoxP3 positive CD3 lymphocytes in all the treated groups compared to group A (1.76% vs. 4.85% vs. 3.015% vs. 4.14 % for groups A, B, C and D respectively).
3.2. Oral administration of Ambroxol with GC was associated with a pro- to an anti-inflammatory cascade shift


Fig. 2A shows that the beneficial effects on the liver damage associated with a pro-inflammatory to anti-inflammatory cytokine shift in the serum. There was a trend for decrease in the level of serum IL-1a in the treated groups as compared to the control group A (206.5 vs. 48 vs. 131 vs. 42 pg/ml for groups A, B, C and D respectively, P = 0.09 for A vs. D). However, the decrease in group B and C compared to group A wasn’t statistically significant (P = 0.11 for group A vs. B and P = 0.63 for group A vs. C).
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Fig. 2. Effects of Ambroxol and GC on serum cytokine profile. Serum cytokines were measured at the end of the study for IL-1a (A); IL-6 (B); IFN-y (C); IL-12 (D); IL-4 (E).

Similarly, Fig. 2B showed a significant decrease of serum IL-6 in the treated group (11768 vs. 984 vs. 9504 vs. 567 pg/ml for groups A, B, C and D respectively, P < 0.0001 for group D vs. A, P = 0.98 for group D vs. B and P < 0.0001 for group D vs. C). However, the decrease in group C compared to group A didn’t reach a statistical significance (P = 0.38).

Fig. 2C showed a trend for decrease in the pro-inflammatory cytokine INF-γ in groups, B and D compared to group A (36 and 19 vs. 643 pg/ml, respectively, P = 0.07 and P = 0.06 for group B and D compared to group A). Similarly, there was a statistically significant decrease in group D compared to C (P = 0.01). However, there was no decrease in INF-g in group C compared to group A (P = 0.99).

Fig. 2D showed the effects on the serum level of IL-12. There was a decrease in all the treated groups as compared to the control group A. The difference was statistically significant for group A vs. B (P = 0.03), however the change between the other groups didn’t reach statistical significance (P = 0.53 for C vs. A, P = 0.34 for group D vs. B and P = 0.91 for group C vs. D).

Fig. 2E showed a trend for increase in the serum level of the anti-inflammatory cytokine IL-4, mainly in group D (32 pg/ml) as compared to group A (22 pg/ml), group B (8.4 pg/ml) and group C (8.7 pg/ml). The difference didn’t reach statistical significance (P = 0.1 for group D vs. B and group D vs. C).

No statistically significant effects were noted in the serum levels of IL10, Il-17, and TGF-β (data were not shown).
3.3. Oral administration of Ambroxol and GC alleviated immune-mediated liver injury


Fig. 3 shows the effect of oral administration of the either the Ambroxol, GC, or a combination of Ambroxol and GC on the immune-mediated liver injury as measured by the effect on liver enzymes (ALT and AST). The oral administration of the combine treatment of Ambroxol and GC was associated with a significant alleviation of the liver injury compared to the untreated controls in group A (24040 vs. 4198 vs. 16455 vs. 2235 IU for ALT levels; and 9140 vs. 2939 vs. 8516 vs. 1470 IU for AST levels, for groups A, B, C, and D, respectively. For ALT, P = 0.005 for group B vs. A, P = 0.003 for group D vs. A, and P = 0.01 for group D vs. C. For AST, P = 0.01 for group B vs. A, P = 0.004 for group D vs. A, and P = 0.001 for group D vs. C.
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Fig. 3. Effect of oral administration of Ambroxol and GC on liver enzymes. There was a significant decrease in liver enzymes (ALT and AST) in all treated groups.

Notably, the decrease in the liver enzymes was more profound in group D compared to group B, however, the difference didn’t reach any statistical significance (P = 0.94 for ALT and P = 0.7 for AST). Moreover, the decrease in group C wasn’t statistically significant compared to group A.


Fig. 4 shows the representative sections from the liver biopsies performed at the end of the treatment period. The Alleviation of the liver apoptosis and improved hepatocyte architecture were noted in the mice in all the treated groups compared to the untreated controls
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Fig. 4. Effects of Ambroxol and GC on liver histology at the end of the study. Hematoxylin Eosin staining was performed on liver sections from treated and control mice. Amelioration of hepatocytes apoptosis coupled with improved liver architecture was noted in treated groups B, C and D compared with control group A.
4. Discussion

The results of the present study show that the co-administration of Ambroxol and GC exert a synergistic effect on the immune system, alleviating the immune-mediated liver damage in a ConA hepatitis model. A significant synergistic effect on the suppression of intrahepatic CD8+CD25+, which increase in the CD8 lymphocyte trapping, and increase in the serum IL4 levels was shown for the Ambroxol with the GC-treated group.

CD8+CD25+ lymphocytes exert immunosuppressive functions in various organs both in the animal models and humans [30,31]. This subset of cells exerts an atheroprotective effect in experimental atherosclerosis [32]. The manipulation of these cells prevented chronic allergic inflammation and improve lung function in allergic asthma exacerbation [33]. The data of the present study support the redistribution of this subset of regulatory lymphocytes, as part of the overall regulatory immunomodulation exerted by the treatment. The increase in the FoxP3 cells further supports the promotion of regulatory lymphocytes in the treated groups.

A synergistic effect of the combination of Ambroxol with GC was noted for the CD4/CD8 lymphocyte ratio. The CD4/CD8 ratio in the spleen increased in the combination-treated group compared with other groups. Interestingly, the intrasplenic/ intrahepatic ratio of the CD4/CD8 ratios was markedly increased only in the combination-treated group D compared to all other groups suggesting a sequestration of CD8+ cells in the liver. Intrahepatic lymphocyte trapping was described as a mechanism for the immune regulation [15,17,[34], [35], [36], [37], [38]]. Antigen presentation by the liver cells controls intrahepatic T cell trapping [39]. In the immune-mediated colitis model, the liver is an important site for CD8+ accumulation during tolerance induction [34]. A similar effect was shown in the models of fatty liver disease and diabetes [40].

A synergistic effect of the combination of Ambroxol and GC was also noted for the increase in the serum IL4 levels. While IL4 was suggested to be associated with the ConA liver damage [41,42], other studies show that its role may be somehow diverse [43]. An altered pro to anti-inflammatory cytokine balance, or different types of effects on the NKT cells which are associated with the IL4 secretion in this model, may partially explain these differences [16,17,[43], [44], [45]]. An altered distribution of the NK1.1 cells and suppression of pro-inflammatory cytokines, IL1a, IFNγ, IL6, and IL12 was shown in the treated groups, further supporting an anti-inflammatory cytokine shift. These effects on the immune system were associated with marked alleviation of the liver damage demonstrated by a decrease in the liver enzymes and amelioration of the histological liver damage.

Ambroxol hydrochloride has been extensively investigated in pulmonary diseases and is mostly prescribed as a mucolytic therapy. In an LPS-mediated acute lung injury model, Ambroxol alleviated lung inflammation and decreased IL-6, TNF-α, and TGF-β pro-inflammatory cytokines secretion [46]. Ambroxol was shown to exert an anti-oxidative and anti-inflammatory effect in vitro and in vivo [[47], [48], [49]], and an organ preservation effect in a transplantation model [50].

Glycosphingolipids are being explored as secondary messengers in various systems [21,24,51]. Their protective immune effect was described both in animal models and humans [13,15,18,20,23,45,[52], [53], [54]]. GC serves as a potent immune adjuvant in the gut for oral immunotherapy [[55], [56], [57], [58]]. Part of the beneficial effect of the GC is associated with the alteration of the NKT-mediated immune balance, and by the alteration of the NKT-dendritic cells cross talk [13,19,23,44]. The patients with Gaucher disease have high levels of GC. Both the clinical and laboratory data suggested a potential beneficial effect for GC in these [59].

Recently the combination of Ambroxol with GC was suggested as a possible mode of therapy for the patients with Parkinson's disease [60]. The patients with non-neuronopathic Gaucher disease and heterozygous GBA mutation carrier are at increased risk for Parkinson disease [61,62]. However, the risk of Parkinson does not increase with accumulation of GC, and is not higher in heterozygous GBA mutation carrier relative to Gaucher patients with two mutated alleles [63]. This observation suggests that the risk for PD is not correlated with the acid beta-glucocerebrosidase (GCase) activity. It further supports that extracellular GC may have beneficial anti-inflammatory properties. Based on these, administration of GC to GBA carriers at risk for PD was suggested as a means to slow inflammatory-driven secondary neuronal death.

Ambroxol is a pharmacological chaperone which reduces endoplasmic reticulum stress induced by the accumulation of mutant misfold GCase. It may prevent the primary pathologic process that leads to cell death [9,64]. GC may act synergistically with Ambroxol in GBA carriers [60]. The present study further supports a synergism of GC and Ambroxol.

Immunosuppression is a major obstacle for long-term use of most immunomodulatory agents including biologicals [65]. Both the Amobroxol and GC have a high safety profile. The data of the present study suggest that their co-administration may provide a mean for a safe immunomodulation which does not induce immunosuppression. Further studies are required to test the effect of therapy on functional assays on CD8+ and CD4 + T cells, as well to elaborate on the potential effect of therapy on macrophages and dendritic cells and on liver NK1.1 and CD4+Foxp3 + .

In summary, the co-administration of the Ambroxol with GC exerted a synergistic immunoprotective effect in a model of the immune-mediated acute liver damage. Bearing in mind the high safety profile of both agents, this combination may become a novel immunomodulatory non-immunosuppressive therapeutic platform.
Authorship Contributions

Tawfik Khoury, Devorah Rotnemer-Golinkin, Lidya Zolotarovya, Yuval Ishay: conducted the studies, and analyzed the data.

David Arkadir, Ari Zimran, Yaron Ilan: Designed the study and wrote the manuscript.

All authors reviewed the final version.
Declaration of Competing Interest

The authors report no declarations of interest.
Acknowledgments

NA.

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