Fluconazole worsened lung inflammation, partly through lung microbiome dysbiosis in mice with ovalbumin-induced asthma

Animal and animal model

The animal study protocol received approval from the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University, under NIH guidelines (SST 016/2566). To avoid the influence of estrogen on asthma, male C57BL/6 8-week-old mice (20–25 g) were obtained from Nomura Siam International, Pathumwan, Bangkok, Thailand. Briefly, all mice were housed in a specific pathogen-free (SPF) animal research facility with a controlled environment, including a temperature of 24 ± 2 °C, 50% relative humidity, and a 12-h light-dark cycle, with light provided from 7:00 a.m. to 7:00 p.m. Six mice were housed in cages with corncob bedding, which was autoclaved before use. The mice had continuous access to a standard sterile diet and autoclaved tap water, provided via a feeder and a permanently placed bottle within the cage. All mice were randomly divided into four groups, each containing six mice, which included: (i) the normal control (Control), (ii) OVA-induced asthma (Asthma), (iii) Fluconazole administration (Antifungal control), and (iv) the Fluconazole-treated OVA-induced asthma group. Six mice in each group were randomly divided into two subgroups of three mice each, and the experiments were independently repeated twice. The OVA-induced asthma mice were given a combination of 50 µL of Ovalbumin (Sigma-Aldrich, St. Louis, MA, USA) and 1.6 mg of Alum Adjuvant (aluminum hydroxide; Thermo Scientific, Rockford, IL, USA) through intraperitoneal injection (i.p.) on day 0 and day 7 before the sensitization by intratracheal (i.t.) administration of a 50 µg OVA solution on days 14, 21, and 22. To investigate the antifungal treatment, a daily oral administration of 250 µL of a 2 mg/mL fluconazole solution was performed for 1 week as a previous publication (Heng, Jiang & Chu, 2021). This was conducted in groups receiving fluconazole alone and asthma with fluconazole. Twenty-four hours after the final dose of fluconazole, all mice were humanely euthanized via cardiac puncture using an overdose of isoflurane (Abbott Laboratories Ltd., Bangkok, Thailand). Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for analysis according to the experimental schema (Fig. 1A).

Mouse sample analysis

Serum cytokines (IL-4, TNF-α, IL-6, and IL-10) and IgE were measured by the enzyme-linked immunosorbent assay (ELISA) (Invitrogen, Waltham, MA, USA). Additionally, lungs (0.1 g) were homogenized through sonication (20 s of pulse-on, 5 s of pulse-off on ice for a total of 45 s) before centrifugation and evaluated tissue cytokines (IL-4, TNF-α, IL-6, and IL-10) and IgE using ELISA (Invitrogen). Fungal identification in lung samples was performed via culture and PCR, following established protocols (Khot & Fredricks, 2009). Briefly, mouse lung tissue was homogenized by a lysis buffer at 65 °C for 3 h, digested (mechanical bead beater for 20 min at 15 vibrations per second) before the extraction of metagenomic DNA by Phenol: Chloroform method, and amplified by the universal Internal Transcribed Spacer (ITS) eukaryotic primers; ITS3 (forward: 5′-GCATCGATGAAGAACGCAGC-3′) and ITS4 (reverse: 5′-TCCTCCGCTTATTGATATGC-3′). Furthermore, the lung microbiome analysis was performed according to the previous protocol (Toju et al., 2012). Briefly, lung tissue (0.1 g) was used for purifying metagenomic DNA as a template for amplifying the V3-V4 region of the 16S rRNA-encoding gene. It was sequenced by the Illumina Miseq sequencing platform (Illumina, San Diego, CA, USA). Universal prokaryotic 16S rRNA primers, 515F (forward: 5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (reverse: 5′-GGACTACHVGGGTWTCTAAT-3′), supplemented with Illumina adapter and Golay barcode sequences, were employed to construct the bacteriome sequencing library for the 16S rRNA gene. The detected mouse lung 16S rRNA sequences were deposited in the NCBI database (accession number PRJNA1019875).

The histology, the lung tissue was preserved in a 10% paraformaldehyde solution, followed by embedding in paraffin and sectioning at a 5 µM thickness before staining by Hematoxylin and eosin (H&E) or Masson’s Trichrome colors for pulmonary morphology and lung fibrosis, respectively, following standard protocol (Udompornpitak et al., 2023). The histological analysis was conducted by two blinded analysts in 10 randomly selected areas per slide based on immune cell infiltration, alveolar thickening, and parabronchial infiltration with the following scores: 0 (indicating no discernible findings) to 4 (indicative of diffuse inflammation with over 50 visualized lumens), as previously published (Phuengmaung et al., 2022).

For the collection of BALF, mice were euthanized with an overdose of isoflurane following a previously published protocol (Kalidhindi, Ambhore & Sathish, 2021). In short, a small horizontal incision was made in the trachea using a sharp scalpel, then 1× phosphate-buffered saline (PBS), pH 7.4 (1 mL), was slowly injected into the cannula and the injected PBS back into the syringe, and repeated the process with an additional 1 mL of PBS. The BALF was centrifuged at 2,000 × g for 5 min at 4 °C and the cell pellets were resuspended in 1 mL of PBS and were stained with Wright-Giemsa color (Sigma-Aldrich) using a standard protocol (Tongthong et al., 2023). The immune cells (mononuclear cells, eosinophils, and neutrophils) were subsequently analyzed by two blinded pathologists in 10 randomly selected fields (×400 magnification) per slide.

The in vitro experiments

To explore the impacts of fungal and bacterial components on the human pulmonary mucoepidermoid carcinoma cell line (H292) (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) from Gibco, Carlsbad, CA, USA, and 1% PenStrep. The cell culture was maintained at 37 °C in a 5% CO₂ incubator. Subsequently, H292 cells at a concentration of 1 × 10⁶ cells per well were incubated with lipopolysaccharides (LPS) (Escherichia coli 026: B6; Sigma-Aldrich, St. Louis, MA, USA) (a representative of Gram-negative bacterial cell wall) (100 ng/mL) with or without (1→3)-β-D-glucan (BG), a representative fungal cell wall component, at concentrations of 0.1 or 1 mg/mL at 37 °C in a 5% CO₂ environment. Cytokines (TNF-α, IL-6, IL-8, IL-10) in supernatant samples were measured using ELISA (Invitrogen).

Statistical analysis

Data were presented as mean ± standard error (SE) using SPSS 11.5 and GraphPad Prism version 7.0 for the statistical analysis. One-way analysis of variance (ANOVA) followed by Tukey’s analysis and Student’s t-test was used for multiple groups and two group comparisons, respectively. A p-value of less than 0.05 was considered statistically significant.

Fluconazole worsened lung inflammation in OVA-induced asthmatic mice

The experimental setup for in vivo test is illustrated in Fig. 1A. Asthmatic mice with and without fluconazole exhibited retardation of weight gain (Fig. 1B), increased IgE and IL-4 in serum (Figs. 1C and 1D), increased serum IL-6 but not serum TNF-α (increased serum IL-10 only in asthma with fluconazole) (Figs. 1E–1G), and prominent IL-4 and IgE in the lung homogenates (Figs. 1H and 1I). For the local lung inflammation, lung TNF-α and IL-6, but not IL-10, in the OVA-fluconazole group was higher than the OVA mice (Figs. 1J–1L), while OVA mice demonstrated increased fungal burden in the lungs, as indicated by elevated ITS gene expression, a marker of fungal DNA (Fig. 1M). Histological analysis revealed greater lung damage score and prominent monocytes in bronchoalveolar lavage fluid (BALF), but not neutrophil nor eosinophil, in fluconazole-treated asthmatic mice compared with the OVA asthma group (Figs. 1N–1O and Fig. 2). Notably, the more prominent mononuclear cell infiltration and subepithelial fibrosis in fluconazole-treated asthma over asthma alone was indicated in the Hematoxylin and Eosin (Fig. 2 upper) and Masson’s Trichrome (Fig. 2 lower) colors, respectively. However, fluconazole-induced asthma and asthma alone exhibited similar parameters, including lung inflammatory cytokines, mononuclear cells in bronchoalveolar lavage fluid (BALF), and lung damage scores (Figs. 1J–1L, 1N, 1O). Notably, an increase of immune cells was observed in the lungs of asthma mice (Fig. 1N), which may contribute to the elevated levels of TNF-α and IL-6 in the lung tissue. These findings support the characterization of asthma as a local inflammatory disease (Lemanske, 2000).

Lung microbiota alteration in asthma and impacts of fluconazole

Because the local inflammatory responses in OVA-administered mice might partly be due to the alteration of lung microbiota from the induction of asthmatic responses, microbiome analysis from the lung tissue (the lower respiratory tract) was performed, as such, the relative abundance of bacteria in different levels, including the class, order, and family (Fig. S1), and the abundance in the genus (graph and heat map presentations) (Figs. 3A, 3B) together with the beta diversity, using Bray-Curtis distance and Generalized UniFrac (GUniFrac) with an alpha of 0.5 (similarity separation) (Fig. 4A), and the determination of representative bacteria in each group (the linear discriminant analysis; LDA) with the alpha diversity (Figs. 4B, 4C) were performed. For bacterial abundance, Proteobacteria (mostly Gram-negative aerobic pathogenic bacteria) was dominant in all groups (non-different among groups), followed by Firmicutes (mostly Gram-positive anaerobic beneficial bacteria) (Fig. S1, phylum level).

In fluconazole-treated asthma, there appears to be a lower abundance of Firmicutes than in non-treated asthma and a higher abundance of Bacteroidota (former name was Bacteroidetes; mostly Gram-negative anaerobic bacteria) when compared with fluconazole-treated control (Figs. 5A, 5B and Fig. S1, phylum level). The increased Firmicutes in the Fluconazole group compared to the control, along with the reduced fungi (Fig. 1M), support the impacts of fungi on bacterial population (Medeiros et al., 2023). Surprisingly, there seems to be no difference in the abundance of Proteobacteria in the lungs between the control and asthma groups (Fig. 5C). The increased Bacteroidota with decreased Firmicutes in fluconazole-asthma (Fig. 5B) might elevate immune responses against Bacteroidota (Gram-negative bacteria) because of the potent innate immune activation of LPS (the major component of Gram-negative bacterial cell wall) (Alexander & Rietschel, 2001), that possibly worsen the severity of asthma. However, the LDA score indicated E. coli-Shigella as the representative bacteria in mice with fluconazole alone, and several Desulfobacteria represented the asthma control group without the representative bacteria in other groups (Fig. 4C).

Although the bacterial abundance among groups was only subtly different, the beta and alpha diversities (Figs. 4A and 4B) indicated notable differences. For beta diversity (an alteration in bacterial species), the principal coordinate analysis (PCoA) based on Bray Curtis dissimilarity metrics (a simplified method to demonstrate the similarity among groups using the 2-dimensional distances from the axis) indicated the similarity among normal control mice (green dots) (Fig. 4A, upper part). There was a clear difference between i) control and fluconazole alone and ii) control and fluconazole-asthma but not between control and asthma alone, as calculated by the GUniFrac with alpha 0.5 (Fig. 4A, lower part; distance to control and distance to fluconazole), showed that microbiota communities were separated among the sample groups (PERMANOVA test; p = 0.03796). However, other beta-diversity plots showed no significant difference among the groups. In parallel, the alpha diversity (the species diversity or richness of the species within the community), using observed operational taxonomic unit (OTUs), Chao-1, and Shannon score, indicated the highest bacterial diversity in the control group, while fluconazole-asthma indicated the lowest diversity (Fig. 4B). There was no statistically significant difference among the sample groups (Kruskal Wallis test; p > 0.05) (Fig. 5B). These data indicated a subtle change in lung microbiome between control and asthma but a more prominent alteration with the use of fluconazole (Figs. 4A–4C) with the direction toward reduced Firmicutes and increased Bacteroides (Figs. 5A, 5B).

The pro-inflammatory impact on lung cells with the presence of Lipopolysaccharide and β-glucan

Due to the elevated fungi from asthma (Fig. 1M), perhaps through the transfer of oropharyngeal Candida into the lung during respiratory distress (Gani et al., 2020), there seems to be an increase in Bacteroides in the fluconazole-asthma group (Fig. 5B), the co-presentation of LPS and β-glucan (BG), the major cell wall components of Gram-negative bacteria and fungi, respectively, are possible. In comparison with LPS alone, the additive pro-inflammatory effect of LPS and BG (1 mg/mL) on H292 cells (the pulmonary cell line) was demonstrated by TNF-α and IL-6 but not on IL-8 and IL-10 (Figs. 6A–6D). The highest TNF-α production was shown in LPS plus high dose BG (1 mg/mL) (Fig. 6A), while BG alone activated only TNF-α but not IL-6 (Figs. 6A, 6B). All stimulations had no response with IL-8 and IL-10 (Figs. 6C, 6D). These data suggest a potential additive pro-inflammatory effect of fungi and bacteria, potentially leading to heightened pulmonary cell inflammation and the worsening of severe asthma.