Signals from intestinal microbiota mediate the crosstalk between the lung-gut axis in an influenza infection mouse model

Abstract

Introduction: Introduction: The influenza virus primarily targets the respiratory tract, yet both the respiratory and intestinal systems suffer damage during infection. The connection between lung and intestinal damage remains unclear.

Methods: Our experiment employs 16S rRNA technology and Liquid Chromatography-Mass Spectrometry (LC-MS) to detect the impact of influenza virus infection on the fecal content and metabolites in mice. Additionally, it investigates the effect of influenza virus infection on intestinal damage and its underlying mechanisms through HE staining, Western blot, Q-PCR, and flow cytometry.

Results: Our study found that influenza virus infection caused significant damage to both the lungs and intestines, with the virus detected exclusively in the lungs. Antibiotic treatment worsened the severity of lung and intestinal damage. Moreover, mRNA levels of Toll-like receptor 7 (TLR7) and Interferon-b (IFN-b) significantly increased in the lungs post-infection. Analysis of intestinal microbiota revealed notable shifts in composition after influenza infection, including increased Enterobacteriaceae and decreased Lactobacillaceae. Conversely, antibiotic treatment reduced microbial diversity, notably affecting Firmicutes, Proteobacteria, and Bacteroidetes. Metabolomics showed altered amino acid metabolism pathways due to influenza infection and antibiotics. Abnormal expression of indoleamine 2,3-dioxygenase 1 (IDO1) in the colon disrupted the balance between helper T17 cells (Th17) and regulatory T cells (Treg cells) in the intestine. Mice infected with the influenza virus and supplemented with tryptophan and Lactobacillus showed reduced lung and intestinal damage, decreased Enterobacteriaceae levels in the intestine, and decreased IDO1 activity.

Discussion: Overall, influenza infection caused damage to lung and intestinal tissues, disrupted intestinal microbiota and metabolites, and affected Th17/Treg balance. Antibiotic treatment exacerbated these effects. Supplementation with tryptophan and Lactobacillus improved lung and intestinal health, highlighting a new understanding of the lung-intestine connection in influenza-induced intestinal disease.

Keywords: Lactobacillus; gut-lung axis; influenza A virus; intestinal microbiota; tryptophan metabolism.

Figure 1

Influenza virus infection causes lung and intestinal immune injury. (A) The levels of the influenza virus–derived matrix protein gene in the lungs, colon, and ileum were detected by PCR; (B) Histology analysis including H&E (scale: 100 μm) of the lungs, colon, and ileum was observed at 9 days post infection; (C) Spider web plot displaying histopathological scoring of lung damage; (D) Spider web plot displaying histopathological scoring of colon damage; (E) Spider web plot displaying histopathological scoring of ileum damage. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with influenza virus infection; Ant group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with a combination of antibiotics.

Figure 2

Increased viral RNA, TLR7 signaling, and IFN-α and IFN-β mRNA expression in the lungs, colon, ileum, and their correlation. (A) TLR7 mRNA level; (B) TLR9 mRNA level; (C) Myd88 mRNA level; (D) IFN-α mRNA level; (E) IFN-β mRNA level in the lungs and (F) TLR7 mRNA level; (G) TLR9 mRNA level; (H) Myd88 mRNA level in the colon; (I) TLR7 mRNA level; (J) TLR9 mRNA level; (K) Myd88 mRNA level in the ileum. (L) Expression of TLR7 correlated with viral RNA and IFN-β mRNA levels in the lungs of the virus- and antibiotic-treated mice. Each dot represents data from one animal. Data are mean ± SEM. n = 3. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with influenza virus infection; Ant group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with combination of antibiotics. Statistical analysis was performed by independent samples T test. **P < 0.01; ****P < 0.0001. All replicates are biological.

Figure 3

H1N1 influenza virus-induced IFN-β alters the fecal microbiota diversity and composition; analysis of the fecal microbiota from the control, virus-infected mice, and antibiotic-treated mice during influenza infection. (A) The diversity of fecal microbiota from experimental groups on the 9th day post infection (dpi) was analyzed by sequencing; (B) Graph is the average relative abundance of each bacterial phylum; (C, D) LDA score about control and virus infection group, control, and antibiotic-treated group. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with influenza virus infection; Ant group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with combination of antibiotics.

Figure 4

Analysis of fecal metabolic characteristics associated with H1N1 influenza virus infection by GC/TOF-MS. (A) Fecal matter from mice treated with H1N1 influenza virus or antibiotics for 9 days or treated with PBS were analyzed by GC/TOF-MS and then further examined through partial least squares discriminant analysis (PLS-DA) to compare between the three groups. The 563 metabolic features were examined for pathway enrichment analysis using Metaboanalyst software; (B) The metabolic pathways significantly affected by influenza virus infection between the control and virus-infected groups. (C) The metabolic pathways significantly affected by influenza virus infection and antibiotic treatment between the control and antibiotic-treated groups.

Figure 5

Correlation analysis of intestinal microorganisms and metabolites between the virus-infected group and antibiotic-treated group. (A) virus-infected group; (B) antibiotic-treatment group.

Figure 6

The expression of IDO1, RORγ, and Foxp3 in the colon and ileum. The expression of IDO1 at the transcriptional level and protein level in the (A–C) colon and (D–F) ileum of the experimental group at the 9th day post infection; RORγ and Foxp3 were detected by immunoblot in the (G–I) colon and (J–L) ileum of the experimental group at 9th day post infection. Each dot represents data from one animal. Data are mean ± SEM. n = 3. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with influenza virus infection; Ant group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with the combination of antibiotics. Statistical analysis was performed by independent samples T test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference. All replicates are biological.

Figure 7

Effects of influenza virus infection on the quantity of Treg cells and Th17 cells in the colon and ileum of mice. The frequencies of Treg cells (A), and Th17 cells (B) of colon lymphocytes were analyzed by flow cytometry. The frequencies of Treg cell (C), and Th17 cells (D) of ileum lymphocytes were analyzed by flow cytometry. Treg cell, CD4+Foxp3+; Th17 cell, CD4+Foxp3-RORγt+. Each dot represents data from one animal. Data are mean ± SEM. n = 3. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with influenza virus infection; Ant group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with combination of antibiotics. Statistical analysis was performed by independent samples T test. *P < 0.05; **P < 0.01. All replicates are biological.

Figure 8

Effects of the influenza virus, tryptophann and Lactobacillus on lung and intestinal tissue structure. (A) Histology analysis including H&E (scale: 100 μm) of the lungs, colon, and ileum was observed at 9 days post infection; (B) Spider web plot displaying histopathological scoring of lung damage; (C) Spider web plot displaying histopathological scoring of colon damage; (D) Spider web plot displaying histopathological scoring of ileum damage. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were mice treated with influenza virus infection; Trp group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous tryptophan supplement; Lac group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous Lactobacillus supplement.

Figure 9

Effects of influenza virus, tryptophan and Lactobacillus supplement on the expression of (A) Enterobacteriaceae and (B) Lactobacillus in the intestine. Each dot represents data from one animal. Data are mean ± SEM. n = 3. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were mice treated with influenza virus infection; Trp group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous tryptophan supplement; Lac group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous Lactobacillus supplement. Statistical analysis was performed by independent samples T test. *P < 0.05; **P < 0.01. All replicates are biological.

Figure 10

Effects of influenza virus infection and exogenous supplementation of tryptophan and Lactobacillus on the concentration of tryptophan-related metabolites in the intestine of mice. The concentration of (A) Tryptophan; (B) Picolinic acid; (C) Xanthurenic acid; (D) Tryptamine; (E) Kynurenic acid; (F) Kynurenine in the Con, Inf, Try, and Lac groups at 9th day post infection in the cecum microbiota. Each dot represents data from one animal. Data are mean ± SEM. n = 3. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were mice treated with influenza virus infection; Trp group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous tryptophan supplement; Lac group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous Lactobacillus supplement. Statistical analysis was performed by independent samples T test. *P < 0.05; **P < 0.01. All replicates are biological.

Figure 11

The expression of IDO1 in the intestine. (A) Western blot results for IDO1; (B) Statistical results of IDO1 expression levels in four groups. Protein expression = IDO1 protein level / GAPDH protein level. Each dot represents data from one animal. Data are mean ± SEM. n = 3. Con: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with PBS; Inf group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were mice treated with influenza virus infection; Trp group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous tryptophan supplement; Lac group: Female BALB/c mice aged 6 to 7 weeks (20 ± 2 g) were treated with exogenous Lactobacillus supplement. Statistical analysis was performed by independent samples T test. **P < 0.01; ***P < 0.001. All replicates are biological.

Figure 12

Graphical abstract. Infection with H1N1 influenza virus not only damages the lungs of mice but also causes injury to intestinal epithelial cells and crypt atrophy in the intestine. It induces dysbiosis of the intestinal microbiota, leading to decreased diversity and richness of gut microbiota, reduced abundance of Lactobacillus, and inhibition of metabolic products especially related to tryptophan metabolism pathways. This imbalance results in disrupted proportions of Treg cells and Th17 cells in the intestines, impairing intestinal mucosal immune function. Antibiotic treatment did not significantly improve these effects. However, supplementation with exogenous tryptophan or treatment with Lactobacilli significantly improved pathological damage to the lungs and intestines caused by influenza virus infection, restored the balance of Treg cells and Th17 cells, and recovered intestinal mucosal immune function.