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

<div><p>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 <i>Salmonella</i>-induced colitis, further enhancing <i>Salmonella</i> 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 <i>Salmonella</i>-induced colitis and in altering the intestinal microbial balance after influenza infection.</p></div

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

<p>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 <i>Ifnar1</i>^<i>-/-</i> mice on day 0 before infection (n = 6 WT, n = 6 <i>Ifnar1</i>^<i>-/-</i>), and on day 9 after mock (n = 3 WT, n = 3 <i>Ifnar1</i>^<i>-/-</i>) and PR8 infection (n = 3 WT, n = 3 <i>Ifnar1</i>^<i>-/-</i>) 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 <i>Enterobacteriaceae</i> using 16S qPCR. Fecal samples were collected from mice on day 0 before infection (n = 10 WT, n = 8 <i>Ifnar1</i>^<i>-/-</i>) and on day 9 after mock (n = 5 WT, n = 4 <i>Ifnar1</i>^<i>-/-</i>) and PR8 infection (n = 5 WT, n = 4 <i>Ifnar1</i>^<i>-/-</i>). Copy numbers of <i>Enterobacteriaceae</i> 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.</p

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

<p>Page 1 shows the peak in the weight loss observed at 9 days post PR8 infection in WT and <i>Ifnar1</i>^−/− mice (related to Fig. 1).</p> <p>Page 2: WT and <i>Ifnar1</i>^−/− mice were monitored daily until euthanasia at 8 dpi; weight loss is shown (related to Fig. 2B).</p> <p>Page 3 shows the 16S copy number of <i>Enterobacteriaceae</i> other than <i>Salmonella</i>: for each sample, the <i>Salmonella</i> 16S gene copy number was subtracted from the total <i>Enterobacteriaceae</i> 16S gene copy number (related to Fig. 2I). </p> <p>Page 4 shows <i>Ifnβ </i>and<i> Ifnα4</i> transcription<i> </i>in both the lungs and cecum of WT and <i>Ifnar1</i>^−/− mice after pIC non-surgical intratracheal instillation (top panels) or PR8 infection (bottom panels) (related to Fig.4).</p

PR8-induced IFN-Is sensitize the host to <i>S</i>. Typhimurium infection.

<p>A) Schematic representation of the PR8-secondary <i>S</i>. Typhimurium infection model. B) Body weight loss at 8 dpi of WT and <i>Ifnar1</i>^<i>-/-</i> mice in secondarily infected, <i>S</i>. 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 <i>Ifnar1</i>^<i>-/-</i> mice. D, E) <i>S</i>. 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 <i>Salmonella</i> (F) and total <i>Enterobacteriaceae</i> (I) per μl of microbial DNA from colon content determined at 8 dpi. G, H) <i>S</i>. Typhimurium load in MLN and lungs at 72 h after bacterial infection. Each dot represents one mouse, the geometric mean is indicated. <i>P</i> 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 <i>Ifnar1</i>^<i>-/-</i>, PR8+STM = 9 WT and 9 <i>Ifnar1</i>^<i>-/-</i>, STM = 8 WT and 7 <i>Ifnar1</i>^<i>-/-</i>. N of mice used in each group in (C): PR8 = 5 WT and 2 <i>Ifnar1</i>^<i>-/-</i>, PR8+STM = 8 WT and 4 <i>Ifnar1</i>^<i>-/-</i>. N of mice used in each group in (D, E, G, H): PR8+STM = 9–10 WT and 9–10 <i>Ifnar1</i>^<i>-/-</i>, STM = 9–10 WT and 9–10 <i>Ifnar1</i>^<i>-/-</i>. N of mice used in each group in (F, I): PR8+STM = 4 WT and 4 <i>Ifnar1</i>^<i>-/-</i>, STM = 4 WT and 4 <i>Ifnar1</i>^<i>-/-</i>. Abbreviations are as follows: STM, <i>S</i>. Typhimurium.</p

PR8-induced IFN-Is inhibit antimicrobial activity during <i>S</i>. Typhimurium infection.

<p>WT and <i>Ifnar1</i>^<i>-/-</i> mice were infected with PR8 or PBS on day 0, followed by intragastric (i.g.) infection with <i>S</i>. Typhimurium or LB at 5 dpi. A, C, E) <i>Ifnγ</i>, <i>S100A9</i> and <i>Lcn2</i> transcript levels were detected by qPCR in the mouse cecum of WT and <i>Ifnar1</i>^<i>-/-</i> at 8 dpi. G) Ifnγ level was detected by ELISA in the serum of WT and <i>Ifnar1</i>^{<i>–/–</i>}at 8 dpi. B, D) S100A9 and F, H) Lcn2 were detected by immunoblot in the mouse cecum of WT (B, F) and <i>Ifnar1</i>^<i>-/-</i> (D, H) at 8 dpi. Each dot represents one mouse, the geometric mean is indicated. <i>P</i> 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 <i>Ifnar1</i>^<i>-/-</i>, PR8 = 3–5 WT and 3 <i>Ifnar1</i>^<i>-/-</i>, PR8+STM = 7–10 WT and 7–10 <i>Ifnar1</i>^<i>-/-</i>, STM = 7–12 WT and 7–12 <i>Ifnar1</i>^<i>-/-</i>. N of mice used in each group in (B, D): mock = 2 WT and 2 <i>Ifnar1</i>^<i>-/-</i>, PR8 = 2 WT and 2 <i>Ifnar1</i>^<i>-/-</i>, PR8+STM = 3 WT and 3 <i>Ifnar1</i>^<i>-/-</i>, STM = 3 WT and 3 <i>Ifnar1</i>^<i>-/-</i>.. N of mice used in each group in (F, H): mock = 1 WT and 1 <i>Ifnar1</i>^<i>-/-</i>, PR8 = 2 WT and 2 <i>Ifnar1</i>^<i>-/-</i>, PR8+STM = 4 WT and 4 <i>Ifnar1</i>^<i>-/-</i>, STM = 3 WT and 3 <i>Ifnar1</i>^<i>-/-</i>..</p

PR8-induced IFN-Is reduce inflammatory response in the gut during <i>S</i>. Typhimurium infection.

<p>WT and <i>Ifnar1</i>^<i>-/-</i> mice were infected with PR8 or PBS on day 0, followed by i.g. infection with <i>S</i>. Typhimurium or LB at 5 dpi. (A) <i>Il6</i>, (B) <i>Il10</i>, (C) <i>Cxcl2</i>, (D) <i>Muc2</i>, (E) <i>Cxcl10</i> and (F) <i>Mx1</i> transcript levels were detected by qPCR in the cecum of WT and <i>Ifnar1</i>^<i>-/-</i> mice at 8 dpi. Each dot represents one mouse, the geometric mean is indicated. <i>P</i> 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 <i>Ifnar1</i>^<i>-/-</i>, PR8 = 5 WT and 3 <i>Ifnar1</i>^<i>-/-</i>, PR8+STM = 8–10 WT and 8–10 <i>Ifnar1</i>^<i>-/-</i>, STM = 8–10 WT and 8–10 <i>Ifnar1</i>^<i>-/-</i>.</p

Disruption of the CXCL13-CXCR5 axis alters bacterial burden in <i>Cxcr5−/−</i> mice.

<p>Vaginal swabs were collected throughout the course of infection in BALB/c mice given i.p. injections (day −1 and 2, red arrow) of anti-CXCL13 Ab or irrelevant control Ab. Not significant by 2-way repeated measures ANOVA, n = 3/grp. (B) Vaginal swabs were collected throughout the course of infection in <i>Cxcr5−/−</i> and WT mice. *<i>p</i><0.05 by 2-way repeated measures ANOVA, n = 11/grp. Experiments were and repeated 5 times. (C–E) Single cell suspensions of whole GT lymphocytes were stained for flow cytometric identification of neutrophils (Gr-1⁺ & CD11b⁺); cDC (CD11c⁺, CD11b⁺, CD3⁻, CD19⁻ CD40⁺) and activated NKT cells (α-GalCer tetramer, NK1.1⁺, CD3⁺; CD69⁺). Dotplots were gated on singlet lymphocytes and the above markers. Bar graphs show the percent of neutrophils, CD40⁺cDC and CD69⁺NKT over the indicted denominator ± SD in GT. *<i>p</i><0.01 as determined by Bonferroni's t-test, n = 4 pools of two mice/group. (F) Mice were vaginally infected with <i>C. muridarum</i> and lymphocytes were isolated at various times following infection from the whole GT. Cells were stimulated <i>ex vivo</i> with PMA and ionomycin and characterized by flow cytometry. Dotplots were gated on CD3 and CD4. Bars show the mean frequency of Th1, IFNγ⁺ cells per group ± SD of 4 mice/group. *<i>p</i><0.05 as determined by Bonferroni's modified t-test.</p

Disruption of the CXCL13-CXCR5 axis increased UGT pathology.

<p>GTs were harvested 49 days after infection and processed en bloc for paraffin sections. (A) Scatter plot and median of chronic inflammatory scores of BALB/c mice treated with anti-CXCL13 Ab versus an irrelevant control Ab or C56BL/6 WT mice versus <i>Cxcr5−/−</i> mice from hematoxylin and eosin stained slides. *<i>p</i><0.05, Mann-Whitney, n = 3–6 mice or 12 oviducts/group. (B) Photomicrograph of trichrome stained sections from oviducts 49 days after infection of C57BL/6 (WT) or <i>Cxcr5</i>−/− mice. Asterisk (WT) and arrows (<i>Cxcr5−/−</i>) show fibrosis and arrowhead (<i>Cxcr5−/−</i>) depicts a dilated oviduct. Sections are at 20×. (C) Fibrosis scores of C56BL/6 WT mice and <i>Cxcr5−/−</i> mice from trichrome stained slides. *<i>p</i><0.01, Mann-Whitney, n = 6 mice or 12 oviducts/group.</p

Lymphocytes accumulate in the oviducts of <i>Cxcr5</i>−/− mice following infection.

<p>GTs were harvested 49 days after vaginal infection with <i>C. muridarum</i> and used to isolate lymphocytes from the oviducts. (A) Representative dotplots of oviduct lymphocyte subsets stained against surface markers and intracellular cytokines. Cells were gated on CD3⁺CD4⁺ or CD3⁺CD8⁺ cells. (B) The number of lymphocyte subsets as determined in panel B, were compared between groups. Bars equal the mean ± SD. *<i>p</i><0.01 (Bonferroni's modified t test). n = 3–4 oviduct pools/group.</p

<i>C. muridarum</i> activates iNKT & type II NKT cells in vitro and in vivo.

<p>(A) Various dilutions of <i>C. muridarum</i> sonicate or α-Gal was added to cultures of type I NKT cell hybridoma 1.2 and a type II NKT cell hybridoma 1.9. IL-2 was measured in triplicate by ELISA. Experiments were and repeated 2–3 times. (B) Scatter plot of oviduct diameter and fibrosis scores from WT mice and <i>Cxcr5−/−</i> mice obtained from hematoxylin and eosin and trichrome stained slides, respectively. *<i>p</i><0.05, Mann-Whitney, n = 6 mice or 12 oviducts/group. (C) Vaginal swabs were collected throughout the course of infection in WT and <i>Cd1d−/−</i> mice. *<i>p</i><0.01 by 2-way repeated measures, n = 6 mice/group.</p