Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons

Abstract

Human influenza viruses replicate almost exclusively in the respiratory tract, yet infected individuals may also develop gastrointestinal symptoms, such as vomiting and diarrhea. However, the molecular mechanisms remain incompletely defined. Using an influenza mouse model, we found that influenza pulmonary infection can significantly alter the intestinal microbiota profile through a mechanism dependent on type I interferons (IFN-Is). Notably, influenza-induced IFN-Is produced in the lungs promote the depletion of obligate anaerobic bacteria and the enrichment of Proteobacteria in the gut, leading to a "dysbiotic" microenvironment. Additionally, we provide evidence that IFN-Is induced in the lungs during influenza pulmonary infection inhibit the antimicrobial and inflammatory responses in the gut during Salmonella-induced colitis, further enhancing Salmonella intestinal colonization and systemic dissemination. Thus, our studies demonstrate a systemic role for IFN-Is in regulating the host immune response in the gut during Salmonella-induced colitis and in altering the intestinal microbial balance after influenza infection.

Fig 1. PR8-induced IFN-Is alter the fecal microbiota composition, Analysis of fecal microbiota in WT and Ifnar1 ^-/- mice performed by MiSeq and 16S qPCR during influenza infection.

A) Experimental model. Fecal samples were collected from mice on day 0 before infection and on day 9 after mock and PR8 infection. Mice were euthanized at 17 dpi. B, C) The fecal microbiota from WT and Ifnar1 ^-/- mice on day 0 before infection (n = 6 WT, n = 6 Ifnar1 ^-/-), and on day 9 after mock (n = 3 WT, n = 3 Ifnar1 ^-/-) and PR8 infection (n = 3 WT, n = 3 Ifnar1 ^-/-) was analyzed by sequencing using the Illumina MiSeq system. Graphed is the average relative abundance of each bacterial phylum (B) and genus (C); the cut-off abundance level was set at 0.5%. D) Analysis of the fecal Enterobacteriaceae using 16S qPCR. Fecal samples were collected from mice on day 0 before infection (n = 10 WT, n = 8 Ifnar1 ^-/-) and on day 9 after mock (n = 5 WT, n = 4 Ifnar1 ^-/-) and PR8 infection (n = 5 WT, n = 4 Ifnar1 ^-/-). Copy numbers of Enterobacteriaceae per μl of fecal microbial DNA is shown. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by One-Way ANOVA (Bonferroni multiple comparison test). ***p < 0.001; ns, not significant. One representative experiment is shown. Abbreviations are as follows: Uncl., unclassified; uninf, uninfected.

Fig 2. PR8-induced IFN-Is sensitize the host to S. Typhimurium infection.

A) Schematic representation of the PR8-secondary S. Typhimurium infection model. B) Body weight loss at 8 dpi of WT and Ifnar1 ^-/- mice in secondarily infected, S. Typhimurium-only and PR8-only infected mice. C) Lung viral load was measured at 8 dpi by plaque assay in secondarily infected and PR8-only infected WT and Ifnar1 ^-/- mice. D, E) S. Typhimurium load in the colon content at 48 h (7 dpi) and 72 h (8 dpi) after bacterial infection. F, I) 16S copy numbers of Salmonella (F) and total Enterobacteriaceae (I) per μl of microbial DNA from colon content determined at 8 dpi. G, H) S. Typhimurium load in MLN and lungs at 72 h after bacterial infection. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by two-tailed Mann-Whitney test in (B, D, E, F, G, H, I). Non-parametric Kruskal-Wallis test was used in (C). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. Two independent experiments are shown in (B, D, E, G, H). A representative experiment is shown in (C). A representative experiment is shown in (F, I). N of mice used in each group in (B): PR8 = 5 WT and 5 Ifnar1 ^-/-, PR8+STM = 9 WT and 9 Ifnar1 ^-/-, STM = 8 WT and 7 Ifnar1 ^-/-. N of mice used in each group in (C): PR8 = 5 WT and 2 Ifnar1 ^-/-, PR8+STM = 8 WT and 4 Ifnar1 ^-/-. N of mice used in each group in (D, E, G, H): PR8+STM = 9–10 WT and 9–10 Ifnar1 ^-/-, STM = 9–10 WT and 9–10 Ifnar1 ^-/-. N of mice used in each group in (F, I): PR8+STM = 4 WT and 4 Ifnar1 ^-/-, STM = 4 WT and 4 Ifnar1 ^-/-. Abbreviations are as follows: STM, S. Typhimurium.

Fig 3. PR8-induced IFN-Is inhibit antimicrobial activity during S. Typhimurium infection.

WT and Ifnar1 ^-/- mice were infected with PR8 or PBS on day 0, followed by intragastric (i.g.) infection with S. Typhimurium or LB at 5 dpi. A, C, E) Ifnγ, S100A9 and Lcn2 transcript levels were detected by qPCR in the mouse cecum of WT and Ifnar1 ^-/- at 8 dpi. G) Ifnγ level was detected by ELISA in the serum of WT and Ifnar1 ^–/–at 8 dpi. B, D) S100A9 and F, H) Lcn2 were detected by immunoblot in the mouse cecum of WT (B, F) and Ifnar1 ^-/- (D, H) at 8 dpi. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by two-tailed Mann-Whitney test.*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. Two independent experiments are shown in A, C, and E. Three independent experiments are shown in G. One representative experiment is shown in B, D, F and H. N of mice used in each group in (A, C, E, G): mock = 3–4 WT and 3 Ifnar1 ^-/-, PR8 = 3–5 WT and 3 Ifnar1 ^-/-, PR8+STM = 7–10 WT and 7–10 Ifnar1 ^-/-, STM = 7–12 WT and 7–12 Ifnar1 ^-/-. N of mice used in each group in (B, D): mock = 2 WT and 2 Ifnar1 ^-/-, PR8 = 2 WT and 2 Ifnar1 ^-/-, PR8+STM = 3 WT and 3 Ifnar1 ^-/-, STM = 3 WT and 3 Ifnar1 ^-/-.. N of mice used in each group in (F, H): mock = 1 WT and 1 Ifnar1 ^-/-, PR8 = 2 WT and 2 Ifnar1 ^-/-, PR8+STM = 4 WT and 4 Ifnar1 ^-/-, STM = 3 WT and 3 Ifnar1 ^-/-..

Fig 4. PR8-induced IFN-Is reduce inflammatory response in the gut during S. Typhimurium infection.

WT and Ifnar1 ^-/- mice were infected with PR8 or PBS on day 0, followed by i.g. infection with S. Typhimurium or LB at 5 dpi. (A) Il6, (B) Il10, (C) Cxcl2, (D) Muc2, (E) Cxcl10 and (F) Mx1 transcript levels were detected by qPCR in the cecum of WT and Ifnar1 ^-/- mice at 8 dpi. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by two-tailed Mann-Whitney test. *p < 0.05, **p < 0.01; ns, not significant. Two independent experiments are shown. N of mice used in each group in (A, B, C, D, E, F): mock = 3 WT and 3 Ifnar1 ^-/-, PR8 = 5 WT and 3 Ifnar1 ^-/-, PR8+STM = 8–10 WT and 8–10 Ifnar1 ^-/-, STM = 8–10 WT and 8–10 Ifnar1 ^-/-.

Fig 5. PR8-induced IFN-Is reduce intestinal pathology during S. Typhimurium infection.

Cecal histopathology of WT (A, B and C) and Ifnar1 ^-/- (A, C and, D) mice. Blinded histopathology scores of cecal samples collected at 8 dpi from PR8- and mock-infected WT and Ifnar1 ^-/- mice, administered or not with S. Typhimurium at 5 dpi. The score of individual mice (circles) and the geometric mean for each group (bars) are indicated in (A). P values were calculated by two-tailed Mann-Whitney test. **p < 0.01; ns, not significant. Two independent experiments are shown. N of mice used in each group in (A, C): mock = 3 WT and 3 Ifnar1 ^-/-, PR8 = 5 WT and 3 Ifnar1 ^-/-, PR8+STM = 7 WT and 7 Ifnar1 ^-/-, STM = 7 WT and 7 Ifnar1 ^-/-. A detailed scoring for the animals shown in (A) is provided; each stacked column represents an individual mouse in (C). B, D) Hematoxylin and eosin (H&E)-stained sections from representative animals for each group in WT (B) and Ifnar1 ^-/- (D) mice. An image at lower magnification (10X) and one at higher magnification (40X) from the same section are shown. Abbreviations are as follows: L, lumen; M, mucosa; SM, submucosa.