High-fat diet impairs microbial metabolite production and aggravates influenza A infection

Abstract

Background: Alterations in the gut microbiom can significantly impact various regions in the human body, including the pulmonary tract. This study investigates alterations in the gut microbiome during a high-fat diet (HFD), particularly short-chain fatty acids (SCFAs), and how these metabolites affect lung infection caused by Influenza A virus (IAV).

Methods: We used a HFD-mouse model to evaluate gut microbiota composition, SCFA levels, and pulmonary outcomes following IAV infection. Microbial changes were analyzed via taxonomic and functional profiling and SCFA levels were measured from non-obese and obese serum donors. Ultimately, acetate's effects were tested ex vivo in human precision-cut lung slices (PCLS) and in vitro in pulmonary epithelial cells. Mechanistic studies investigated the involvement of the SCFA receptor free fatty acid receptor 2 (FFAR2) and intracellular antiviral pathways.

Results: Our data indicates an increased Firmicutes/Bacteroidetes ratio of the gut microbiome and an altered carbohydrate metabolism, leading to reduced SCFA production. Infected HFD mice showed increased IAV titers and sustained microbial alterations. Interestingly, acetate demonstrated antiviral effects in both the human PCLS model and pulmonary cells with an reduced viral replication. These effects depended on FFAR2, which also acts as an IAV co-receptor, as acetate treatment led to FFAR2 internalization and influenced host cell metabolism in our in vitro data.

Conclusion: HFD alters the SCFA production, reducing acetate levels in the gut microbiome. This reduction may lead to higher viral loads and worsened disease in HFD mice infected with IAV. Our findings indicate that acetate has antiviral effects during IAV infection in both a human ex vivo lung model and pulmonary epithelial cells. Here, acetate prevents viral entry and affects the cellular metabolic state and antiviral response. Understanding these mechanisms could provide new targets for preventing and treating viral infections in individuals with diet-related health issues.

Keywords: Acetate; FFAR2; Gut-lung-axis; High-fat diet; Influenza A virus; Interferon response; Microbial metabolites; Short-chain fatty acids.

Fig. 1

HFD results in a distinct obese phenotype with a shift in gut microbiome composition. Schematic representation of the in vivo diet-induced obesity (DIO) model with a 12-week HFD and collection of fecal pellets before and after the dietary period (A). The 12-week dietary period increased relative body weight (B) (NFD: n = 21, HFD: n = 20) with higher intra-abdominal adipose tissue mass (NFD: n = 6, HFD: n = 6) and blood glucose levels after the diet (NFD: n = 21, HFD: n = 20) (C). Values in panels B and C are displayed as mean ± SD. Gene correlation after shotgun metagenomics sequencing of fecal pellets shows correlation coefficients pre-diet and post-diet (D). HFD caused a taxonomical shift in gut microbiome composition at grouped phylum (E) and genus (F) levels, displayed as heatmaps with each group (n = 3). Additionally, it altered the functional profile after genetic annotation to the KEGG database level 1 (G) and CaZy level 1 (H), shown as absolute value of “Z” representing distance from raw value to population mean in SD units. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P calculated by 2-way ANOVA Šídák’s multiple comparisons test (C) and Mann-Whitney test (D)

Fig. 2

Elevated virus titers and increased pulmonary inflammation in HFD mice infected with IAV. Schematic overview of intranasal infection of NFD (n = 15) and HFD (n = 14) mice with IAV, including organ harvest and fecal pellet collection after 2 and 21 days (A). Scoring values for NFD and HFD mice across 3 weeks p.i. (B). HFD mice (n = 6) exhibited higher active virus particles in lung tissue at day 2 p.i. compared to NFD mice (n = 3) (C). HFD mice showed altered pulmonary cytokine profiles at acute phase (2 days post infection (dpi), HFD n = 6, NFD n = 3) (D) and long-term (21 dpi, HFD n = 6, NFD n = 6) (E) compared to mock NFD (n = 6) and HFD (n = 6) mice. Values are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, calculated by Kruskal-Wallis test, Dunn’s multiple comparison test (C-E)

Fig. 3

Dysregulated gut microbiome taxonomy and SCFA production in infected HFD mice. Gene correlation analysis after shotgun metagenomics from fecal pellets at 2 and 21 days post infection (dpi) from NFD and HFD depends on diet (A). Changes in microbiota composition during infection depend on diet at the phylum level (B). Heatmaps from LefSe analysis show significantly different genera related to SCFA production between HFD (n = 3) and NFD (n = 3) mice at mock, 2 dpi, and 21 dpi. The heatmap values are standardized Z values for relative abundance (C). Reduced expression of Acetate kinase (EC 2.7.2.1) was significant in mock mice after 12 weeks of diet, shown as relative abundance (NFD, n = 3; HFD, n = 3). *P < 0.05 & *Q < 0.05 calculated via Metastats (D)

Fig. 4

Reduction in virus titer in infected human ex vivo lung slices pre-treated with acetate. Healthy female donor cohorts (according to Supplemental Tab. S3) were characterized by a normal BMI (< 25 (kg/m²), n = 4) and an increased BMI for obese individuals (> 30, n = 6) and elevated systemic leptin levels (A). There was a trend towards reduced systemic acetate (p = 0.2189, 𝑅2 = 0.3376), butyrate (p = 0.6727, 𝑅2 = 0.02703), and propionate (p = 0.4392, 𝑅2 = 0.1009) levels (B). Schematic overview of human ex vivo lung slices (PCLS) treated with SCFAs for 2 days and subsequent infection with IAV for 2 days (C). Acetate treatment of lung slices resulted in lower active virus particles, or plaque forming units (PFU/ml) in supernatants (D). Solely IAV infection increased levels of IFN-α2, IFN-γ, and TNF-α, while acetate pretreatment slightly enhanced cytokine production (E). Reduced IAV matrix protein (green) expression in infected lung slice pre-treated with acetate within the alveolus, co-stained with CD68 (red), DAPI (blue), and Phalloidin (purple) (F). Scale bar = 100 μm. Values in panels A, B, D, and E are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, P calculated by Mann-Whitney test (A, D), unpaired t-test with Welch’s correction (B), one-way ANOVA, Tukey’s test (E)

Fig. 5

Antiviral effect of acetate depends on FFAR2 activation and internalization dynamics in pulmonary epithelial cells. Schematic representation of A549 cells pre-treated with acetate and infected with IAV for 8 and 24 h (A). Data represent n = 9 independent biological replicates per condition. Acetate treatment reduced active virus particles in the supernatant of infected wildtype A549 at 8 and 24 h post infection (hpi) (B). Acetate treatment increased ß-arrestin2 recruitment to the cell membrane (arrows) in HEK293 cells expressing FFAR2 compared to untreated and FFAR2-deficient cells (C). Furthermore, acetate stimulation caused FFAR2 translocation from the plasma membrane (CAAX) to early endosomes (FYVE) (left), while ß-arrestin2 also translocated to early endosomes (right) (D). Reduction of active virus particles was absent in A549 cellswith global FFAR2 receptor knock-out (E). IP-10 levels were increased in response to infection in supernatants of both A549 WT and FFAR2-KO cells, with a noticeable increase in WT cells at 24 hpi with IAV (n = 3) (F). Acetate pre-treatment on A549 with global knock-out of IP-10 did not affect the number of active virus particles after 8 or 24 hpi (n = 9) (G)

Fig. 6

Early antiviral effect of acetate during IAV infection in epithelial cells in vitro. Heatmaps of differentially expressed genes at 8 h post infection (hpi) showed more upregulated IAV response genes in acetate pre-stimulated A549 WT cells compared to controls, except for DDX58 (RIG-1) and IRF9 (A). At 24 hpi, there was again an increase in upregulated genes in infected cells pre-treated with acetate compared to controls, except for CALCOCO2, CXCL8, and JAK2 (B). Importantly, CXCL10 is the gene encoding for IP-10