Ambroxol suppresses influenza-virus proliferation in the mouse airway by increasing antiviral factor levels

Animals and materials

Specific pathogen-free female 3-week-old ddY 19 mice weighing 8–10 g (Japan SLC Inc., Shizuoka, Japan) were used. All animals were treated in accordance with the guidelines for animal experimentation set by Tokushima University. Mouse-adapted influenza A/Aichi/68 (H3N2) virus, a laboratory adaptation of the H3N2 pandemic influenza-virus subtype 20, was propagated in 10-day-old embryonated chicken eggs.

Virus infection and administration of ambroxal

Under anaesthesia with ether, mice were infected intranasally with 6.6×10⁴ plaque forming units (PFU) of influenza-A virus in saline, the administration volume being 20 µL. Within 10–15 min of infection, each group of 10 animals received intraperitoneally (i.p.), twice daily, 200 µL injections of ambroxol (Boehringer Ingelheim, Ingelheim am Rhein, Germany) in saline at a total daily dose of 0, 4, 10, 20, and 30 mg·kg⁻¹ body weight, respectively, for 7–10 days. For analysis of the concentrations of various compounds in the airway fluid and pathological changes, three groups of animals treated with ambroxol at 0, 10 and 30 mg·kg⁻¹·day⁻¹ were selected, 80 mice being used for each group. The virus levels in the airway fluid were examined by the immunofluorescent cell-counting method as reported previously 21.

Preparation of bronchoalveolar lavage fluid

Ambroxol was administrated i.p. to each mouse shortly after infection with influenza virus, and then 2 mL of bronchoalveolar lavage fluid (BALF) from each of five surviving mice was prepared from each group every day for 7 days as reported previously 22. The BALF was stored at −80°C until use.

Surfactant protein A, cytokine and immunoglobulin assays

Since there is 95% identity between the amino acid sequence of the SP-As of mouse and rat 23, 24, and isolated polyclonal antibodies against rat SP-A 6, 25 react with mouse SP-A, the levels of SP-A in BALF of mice were analysed by enzyme-linked immunosorbent assay (ELISA) using monospecific biotinylated and nonbiotinylated antibodies against rat SP-A and mouse SP-A as standards. The levels of cytokines (TNF-α, IL-12, IFN-γ, IL-6 and IL-4) in BALF were determined using ELISA kits (BioSource International, Camarillo, CA, USA) according to the manufacturer's protocol. The levels of IgG and IgA in BALF were also analysed using ELISA kits (Bethyl, TX, USA).

The absorbance at 490 nm or 450 nm was read with an ImmunoMini NJ-2300 multiplate reader (Japan Inter Med Co., Ltd, Tokyo, Japan).

Enzyme and inhibitor assays

The trypsin-type protease activity was analysed as previously described 6, using N-tert-butoxycarbonyl-Gln-Ala-Arg-4-methyl-coumaryl-7-amide as the substrate, which resembles the consensus-cleavage motif of HA 4. The inhibitory activity of MPI which accounts for about 90% of the protease-inhibitor capacity in BALF 26, 27 was analysed as follows: an acid and heat stable MPI was extracted from BALF with 5% (percent volume in volume (v/v)) perchloric acid, and then boiled at 100°C for 10 min. Then the supernatant was centrifuged at 15,000×g for 15 min, adjusted to pH 7.0 with 4 M KOH and the inhibitor activity of the supernatant was analysed as previously described 8.

Statistics

All values are given as mean±sd. Significant differences between the values for groups treated with ambroxol and controls without treatment were assessed using a paired t-test, a value of p<0.05 was considered to be statistically significant.

Amboroxol improves the survival rate of mice infected with influenza virus

The administration of ambroxol itself up to the dose 30 mg·kg⁻¹·day⁻¹ into mice produced no apparent toxicity. After virus infection, significant loss of body weight was evident on day 2, and all animals (n=10) without ambroxol treatment died within 10 days. Amboroxol treatment significantly improved the survival rates of infected mice in a dose-dependent manner, with a peak effect at a dose of 10 mg·kg⁻¹·day⁻¹, but higher doses of ambroxol were less effective (fig. 1⇓). When mice were treated with ambroxol at 10 mg·kg⁻¹·day⁻¹, one-half of them survived a dose of influenza virus, which is normally lethal, at day 10. The body weight of all surviving animals recovered rapidly after 10 days. Ambroxol inhibited virus proliferation. To analyse the mechanism underlying the improvement of the survival rate of infected mice by ambroxol, the virus titres in BALF were measured. After intranasal infection with influenza-A virus, the virus titre in BALF increased markedly after a lag period of 2 days, reaching a peak on day 5, and viral replication ceased on day 7, probably due to the host immunological response (fig. 2a⇓). Treatment with ambroxol at a dose of 10 mg·kg⁻¹·day⁻¹ significantly suppressed viral proliferation, but ambroxol at a dose of 30 mg·kg⁻¹·day⁻¹ was less effective. The pathological changes in the lungs, as macroscopic lung lesions, on day 4 (fig. 2b⇓) were also analysed. Severe extensive lesions in the lungs were observed in the infected mice. Treatment with ambroxol at a dose of 10 mg·kg⁻¹·day⁻¹ restricted the pathological changes but its effect at a dose of 30 mg·kg⁻¹·day⁻¹ was less significant.

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Fig. 1.—

The effect of ambroxol on the survival rate of mice infected with influenza-A virus. Mice were infected with 6.6×10⁴ plaque forming units of influenza A/Aichi/68 virus and then injected with saline (•) or ambroxol i.p. twice daily, at a total daily dose of 4 (▴), 10 (▪), 20 (▵), and 30 (□) mg·kg⁻¹·day⁻¹, respectively.

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Fig. 2.—

a) The effect of ambroxol on virus proliferation in bronchoalveolar lavage fluid. Each group of mice (n=80) were infected with influenza-A virus and then treated with saline (•) or ambroxol at a total daily dose of 10 (▵) and 30 (▴) mg·kg⁻¹·day⁻¹, respectively. CIU: cell infecting unit. Data are presented as mean±sd. **: p<0.01. b) Macroscopic lung lesions in mice on day 4 after infection. 1: mice (n=5) on day 4 without infection; 2: mice infected with influenza virus and treated with saline; 3 and 4: mice infected with influenza virus and treated with ambroxal at 10 mg·kg⁻¹·day⁻¹ and 30 mg·kg⁻¹·day⁻¹ respectively.

Although virus proliferation in BALF of infected mice had terminated on day 7, pathological changes in the lungs continued with mild progression and the animals died within 10 days. To elucidate the role of ambroxol in the improvement of the survival rate and suppression of viral proliferation of infected mice, the effects of ambroxol on various cellular factors in BALF that regulate influenza-virus multiplication, and inflammatory cytokines were analysed.

Effects of ambroxol on the levels of activators and inhibitors of influenza-virus multiplication in the airway fluid

Trypsin-type protease, such as tryptase Clara, is constitutively secreted in BALF at basal levels in uninfected mice and rats 6, 9, and influenza-virus infection increased the levels about 6.4-fold with a peak on day 6 (table 1⇓). Treatment with ambroxol at 10 mg·kg⁻¹·day⁻¹ induced further secretion of the protease from day 1, which reached a peak on day 5. Treatment with ambroxol at 30 mg·kg⁻¹·day⁻¹ further increased the level, with an earlier peak on day 4, after which the level decreased rapidly. Constitutive secretion of trypsin-type protease in mice with sham infection was also, but only slightly, stimulated by ambroxol with a peak on day 4 (table 2⇓).

The effects of ambroxol on the concentrations of antiviral defensive factors in the airway, i.e. SP-A and MPI, are shown in tables 1 and 2⇑⇑. Influenza-virus infection increased the secretion of SP-A and MPI, 6- and 4.4-fold, respectively, with a peak on day 6. Treatment of infected mice with ambroxol at 10 mg·kg⁻¹·day⁻¹ caused rapid increases in the levels of MPI and SP-A on day 1 and then further and gradual increases in the levels with a peak on day 5, the levels being ∼9–10-fold and 8.4-fold the basal levels of SP-A and MPI, respectively, and remaining high until day 7. However, on treatment of mice with ambroxol at 30 mg·kg⁻¹·day⁻¹, these peaks were observed earlier on day 4, followed by their rapid reduction. Mild stimulatory effects of ambroxol on the secretion of SP-A and MPI in mice with sham infection were also observed.

Ambroxol stimulates the secretion of mucosal immunoglobulin-A and -G

The basal level of IgA of uninfected mice was very low at 10.3±6.6 ng·mL⁻¹ and that of IgG, which had probably diffused from the serum, was relatively high at 460±26.2 ng·mL⁻¹ (figs. 3a and 4a⇓⇓). Treatment of mice by sham infection with ambroxol at 10 and 30 mg·kg⁻¹·day⁻¹ stimulated the secretion of IgA with a peak on days 7 and 5, respectively and both levels being about 10-fold basal levels. Treatment with ambroxol at 10 and 30 mg·kg⁻¹·day⁻¹also increased the levels of IgG about 1.2-fold on days 7 and 6, respectively (fig. 4a⇓). When mice were infected with influenza virus, the levels of IgA and IgG in BALF increased markedly after a lag of 1–2 days, and reached peaks with an increase in the IgA level of about 400-fold on day 7 and in IgG level of 11-fold on day 6 (figs. 3b and 4b⇓⇓). Treatment of infected mice with ambroxol at 10 and 30 mg·kg⁻¹·day⁻¹ resulted in further increases in the level of IgA about 600-fold basal level on day 7 and 700-fold on day 5, respectively. Ambroxol at 10 and 30 mg·kg⁻¹·day⁻¹ also stimulated the secretion of IgG moderately in infected mice i.e. about 16-fold on day 6 and 15-fold on day 5, respectively. These results indicate that ambroxol predominantly stimulates release of mucosal IgA induced by infection and also that of IgG moderately.

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Fig. 3.—

The effect of ambroxol on mucosal immunoglobulin (Ig)-A in bronchoalveolar lavage fluid (BALF) of mice with a) sham infection and b) influenza-A virus. □: IgA levels in BALF of mice treated with saline; ▪: ambroxol at 10 mg·kg⁻¹·day⁻¹; └: ambroxol at 30 mg·kg⁻¹·day⁻¹. Data are presented as mean±sd (n=5). *: p<0.05; **: p<0.01. NT: no treatment.

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Fig. 4.—

The effect of ambroxol on mucosal immunoglobulin (Ig)-G in bronchoalveolar lavage fluid (BALF) of mice with a) sham infection and b) influenza-A virus. □: IgG levels in BALF of mice treated with saline; ▪: ambroxol at 10 mg·kg⁻¹·day⁻¹; └: ambroxol at 30 mg·kg⁻¹·day⁻¹. Data are presented as mean±sd (n=5). *: p<0.05. NT: no treatment.

Effect of ambroxol on cytokine release

All cytokines examined in BALF of mice with sham infection were under the detection limits. Virus infection significantly induced the production of all cytokines examined, except IL-4, in BALF with different time/course patterns. When mice were infected, TNF-α at first increased, with a peak on day 1, followed by a rapid decrease with a second small peak on day 6. The level of IL-6 also rapidly increased after infection on day 1, the level remaining high with a peak on day 5, but then starting to decrease on day 7. The levels of IL-12 and IFN-γ gradually increased with peaks on days 4 and 6, respectively. IL-4 in BALF of infected mice, however, was not detected during infection for 7 days (data not shown). Treatment of infected mice with ambroxol had suppressive effects on the release of TNF-α on days 3–5, IFN-γ on day 1, and IL-12 on day 4, although the effect of ambroxol was not always evident during infection. Treatment with ambroxol, on the contrary, increased the level of IL-6 on days 4 and 6 in BALF of infected mice (table 3⇓).