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

Abstract

Innate immunity in asthma may be influenced by alterations in lung microbiota, potentially affecting disease severity. This study investigates the differences in lung inflammation and microbiome between asthma-ovalbumin (OVA) administered with and without fluconazole treatment in C57BL/6 mice. Additionally, the role of inflammation was examined in an in vitro study using a pulmonary cell line. At 30 days post-OVA administration, allergic asthma mice exhibited increased levels of IgE and IL-4 in serum and lung tissue, higher pathological scores, and elevated eosinophils in bronchoalveolar lavage fluid (BALF) compared to control mice. Asthma inflammation was characterized by elevated serum IL-6, increased lung cytokines (TNF-α, IL-6, IL-10), and higher fungal abundance confirmed by polymerase chain reaction (PCR). Fluconazole-treated asthma mice displayed higher levels of cytokines in serum and lung tissue (TNF-α and IL-6), increased pathological scores, and a higher number of mononuclear cells in BALF, with undetectable fungal levels compared to untreated mice. Lung microbiome analysis revealed similarities between control and asthma mice; however, fluconazole-treated asthma mice exhibited higher Bacteroidota levels, lower Firmicutes, and reduced bacterial abundance. Pro-inflammatory cytokine production was increased in supernatants of the pulmonary cell line (NCI-H292) after co-stimulation with LPS and beta-glucan (BG) compared to LPS alone. Fluconazole treatment in OVA-induced asthma mice exacerbated inflammation, partially due to fungi and Gram-negative bacteria, as demonstrated by LPS+BG-activated pulmonary cells. Therefore, fluconazole should be reserved for treating fungal asthma rather than asthma caused by other etiologies.

Keywords: Asthma; Fungi; Microbiome; Ovalbumin.

Figure 1. Schema of the establishment of allergic asthma model (see Materials and Methods) (A) and characteristics of control, asthma, fluconazole in normal mice, and fluconazole in asthma as indicated by body weight change (B), serum Ig E (C), and serum IL-4 (D), serum inflammatory cytokines (TNF-α, IL-6, and IL-10) (E–G), lung homogenates (IL-4, Ig E, TNF-α, IL-6, IL-10, and fungal abundance) (H–M), immune cells in bronchoalveolar lavage fluid (BALF) (N), and lung damage score (O) are demonstrated, (n = 6/group).

The data are shown as the mean ± SE, #, ##; p < 0.05 vs. Control, and *, **; p < 0.05 vs. Asthma + Fluconazole using ANOVA with Tukey’s analysis. Portions of this figure created with BioRender.com.

Figure 2. Representative lung histological pictures of control, asthma, fluconazole in normal mice, and fluconazole in asthma as stained by Hematoxylin and Eosin color (upper part) and Masson’s Trichrome (lower part) are demonstrated, (n = 6/group).

Figure 3. Microbiome analysis from the lung of control, asthma, fluconazole in normal mice, and fluconazole in asthma as indicated by bacterial abundance in the genus (graph and heat map presentation) (A, B), (n = 3/group).

Figure 4. Microbiome analysis from the lung of control, asthma, fluconazole in normal mice, and fluconazole in asthma as indicated by beta diversity using the principal coordinate analysis (PCoA) based on Bray Curtis dissimilarity (A upper part) with the distance comparisons (A lower part), the alpha diversity, including observed Operational taxonomic unit (OTUs), Chao-1, and Shannon (B), and the linear discriminant analysis (LDA) (C), are demonstrated, (n = 3/group).

The data are the mean ± SE, **; p < 0.05 vs. Control using ANOVA with Tukey’s analysis.

Figure 5. (A–C) Selected bacterial abundance from the lung microbiome analysis of control, asthma, fluconazole in normal mice, and fluconazole in asthma with the graph presentation (from Figs. S1 and 3A) are demonstrated, (n = 3/group).

Figure 6. Characteristics of pulmonary cells (H292) after the activation by lipopolysaccharide (LPS) with or without beta-glucan (BG) in low (0.1 mg/mL) and high (1 mg/mL) doses as indicated by supernatant cytokines (TNF-α, IL-6, IL-8, and IL-10) are demonstrated (data were derived from triplicated independent isolated experiments), (n = 3/groups with three independent repeats).

The data are shown as the mean ± SE, #; p < 0.05 vs. Cell alone, and *; p < 0.05 using ANOVA with Tukey’s analysis.