Research Article

Recurrent infection progressively disables host protection against intestinal inflammation

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Science  22 Dec 2017:
Vol. 358, Issue 6370, eaao5610
DOI: 10.1126/science.aao5610
  • Gut reaction: Food poisoning triggers chronic inflammation.

    Recurrent Salmonella infections progressively induce host Neuraminidase 3 (green) on the surface of enterocytes among duodenal epithelial cells of the small intestine. Epithelial cells are counterstained to also visualize DNA among cell nuclei (blue) and the cytoskeleton component α-tubulin (red). Elevated neuraminidase activity accelerates the molecular aging and clearance of IAP in triggering colitis.

  • Fig. 1 Recurrent ST infection diminishes the abundance and protective role of IAP.

    Wild-type (WT) mice were analyzed during a course of recurrent ST infection (2 × 103 CFU) or uninfected [phosphate-buffered saline (PBS)] at indicated time points (arrows). (A) Body weight (ST, n = 20; PBS, n = 19). (B) Colon length (n = 40 per condition). (C) Diarrhea and stool consistency (ST, n = 19; PBS, n = 13). (D) Fecal blood (ST, n = 19; PBS, n = 13). (E) Intestinal epithelial barrier function (n = 8 per condition) at 20 weeks of age before the fourth infection. FITC, fluorescein isothiocyanate. (F) Rectal prolapse (ST, n = 30; PBS, n = 20) at 32 to 48 weeks of age or 4 to 20 weeks after last ST infection (representative image). (G) AP activity (n = 40 per condition). (H) Immunoblot blot analysis of IAP at 20 weeks of age before the fourth infection (n = 8 per condition). (I) Relative IAP abundance (n = 40 per condition). (J) AP activity ± cIAP (n = 40 per condition). (K) LPS abundance and phosphate released from LPS (n = 8 per condition) at 20 weeks of age. (L) Body weight (n = 10 per condition), colon length (n = 8 per condition), diarrhea (ST, n = 23; PBS, n = 14; ST + cIAP, n = 19; PBS + cIAP, n = 15), fecal blood (ST, n = 23; PBS, n = 14; ST + cIAP, n = 19; PBS + cIAP, n = 15) at 48 weeks of age (20 weeks after last ST infection), and intestinal epithelial barrier function (n = 8 per condition) at 20 weeks of age before the fourth infection. (M) Cytokine mRNA expression (n = 30 per condition). (N) Hematoxylin and eosin (H&E)–stained intestinal tissues at 48 weeks of age (20 weeks after last ST infection). L, intestinal lumen; E, epithelial layer; C, crypt; G, goblet cell; S, submucosa; I, infiltration of leukocytes. Graphs are representative of 16 fields of view (n = 4 per condition). All scale bars, 100 μm. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; Student’s t test (A, B, E, and G to I) or one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (J to N).

  • Fig. 2 Mechanism of IAP regulation during recurrent ST infection.

    (A to C) Pulse-chase analyses of IAP synthesis, trafficking, and cell surface half-life among cultured primary enterocytes isolated from WT mice at 20 weeks of age (before a fourth ST infection). (D and E) In situ localization and intracellular colocalization of IAP in duodenal sections stained with H&E or with fluorescent antibodies to IAP (green) and intracellular compartment proteins (red), including early endosomes (EEA1), lysosomes (LAMP2), trans-Golgi (γ-adaptin), cis-Golgi (Calnuc), or the endoplasmic reticulum (PDI), depicting the percentage of IAP colocalization (yellow). Graphs are representative of 10 fields of view (n = 4 per condition). Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F) Lectin blot of IAP from small intestine. (G) Lectin binding of IAP on cultured enterocytes after cell surface biotinylation. (A to G) WT mice at 20 weeks of age before fourth infection. (A to C and G) n = 6 per condition. (F) n = 8 per condition. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; Student’s t test (A to C and E and F) or one-way ANOVA with Tukey’s multiple comparisons test (G).

  • Fig. 3 Mechanism of IAP regulation by ST3Gal6 sialylation.

    (A) AP activity. (B) IAP protein abundance. (C) LPS abundance and phosphate released from LPS of intestinal content. (D) Lectin blot of IAP from small intestine. (E to G) Pulse-chase of IAP synthesis and trafficking and IAP cell surface half-life among cultured primary enterocytes isolated from uninfected ST3Gal6-deficient mice and WT littermates at 8 to 10 weeks of age. (H and I) In situ localization and intracellular colocalization of IAP in duodenum, depicting the percentage of IAP colocalization (yellow). Graphs are representative of 10 fields of view (n = 4 per genotype). Scale bars, 10 μm. (A to I) St3Gal6-deficient mice and WT littermates at 8 to 10 weeks of age, uninfected. (A to D) n = 8 per condition. (E to G) n = 6 per condition. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; Student’s t test (A to G and I).

  • Fig. 4 ST3Gal6 sialylation of IAP prevents intestinal inflammation.

    Indicated genotypes after ST reinfection (arrows) were analyzed in the absence or presence of cIAP. (A) AP activity (n = 32 per condition). (B) Body weight (n = 10 per condition), colon length (n = 32 per condition), diarrhea (n = 30 per condition), and fecal blood (n = 30 per condition). (C) Intestinal epithelial barrier function. (D) LPS abundance and phosphate released from LPS of intestinal content. (E) Commensal microbiome 16S rDNA in intestinal content (n = 10 per condition). (F) Inflammatory cytokine RNA in colon and small intestine (n = 24 per condition). (G) H&E-stained colon sections. Graphs are representative of 10 fields of view (n = 4 per condition). Scale bars, 100 μm. (C and D) Data were acquired from mice 20 weeks of age before fourth infection. (E to G) Data were acquired from mice 32 weeks of age and 4 weeks after the last infection. (C and D) n = 8 per condition. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; one-way ANOVA with Tukey’s multiple comparisons test (A to G).

  • Fig. 5 Host Neu is induced by TLR4 during recurrent ST infection.

    WT and Tlr4-deficient mice were analyzed after recurrent ST infections (arrows). (A) Neu activity (n = 30 per condition). (B) NEU1 to NEU4 protein abundance in the small intestine. (C) Neu3 mRNA expression in small intestine. (D) In situ localization of NEU3 and IAP in duodenum. Images are representative of 10 fields of view (n = 4 per condition). Scale bars, 50 μm. (E) AP activity (n = 24 per condition). (F) IAP protein abundance. (G) LPS abundance and phosphate released from LPS of intestinal content. (H) Inflammatory cytokine RNA abundance (n = 16 per condition). (I) Intestinal epithelial barrier function. (B, C, F, G, and I) n = 6 per condition. (B, C, F, G, and I) Animals were 20 weeks of age before the fourth infection. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; one-way ANOVA with Tukey’s multiple comparisons test (A to I).

  • Fig. 6 Host Neu is induced by TLR4 and LPS.

    (A) Neu activity in mice at 8 weeks of age before repeated LPS administrations (arrows). (B and C) Neu protein abundance and Neu3 RNA expression in small intestine. (D) In situ localization of NEU3 and IAP in duodenum sections, representative of 10 fields of view (n = 4 per condition). Scale bars, 50 μm. (E) AP activity before repeated LPS administrations (arrows). (F) IAP protein abundance. (G to I) Phosphate released from LPS of intestinal content, inflammatory cytokine RNA abundance, and intestinal epithelial barrier function. (A and E) n = 24 per condition. (B, C, F, G, and I) n = 6 per condition. (H) n = 16 per condition. (B, C, F, G, and I) Mice on day 6 after LPS administration. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; one-way ANOVA with Tukey’s multiple comparisons test (A to I).

  • Fig. 7 Effects of the Neu inhibitor zanamivir on intestinal inflammation.

    WT mice were analyzed at indicated ages before ST reinfection (arrows) in the absence or presence of zanamivir (Zana) (0.5 mg/ml) provided in drinking water immediately after first infection. (A) Neu activity. (B) NEU1 to NEU4 protein abundance in small intestine. (C) AP activity. (D) IAP protein abundance. (E) LPS abundance and phosphate released from LPS of intestinal content. (F) Lectin blotting of IAP protein from small intestine. (G) In situ localization of IAP in duodenum, representative of 10 fields of view (n = 4 per condition). Scale bars, 20 μm. (H) Body weight (n = 10 per condition), colon length (n = 8 per condition), diarrhea (n = 10 per condition) and fecal blood (n = 10 per condition) at 32 weeks of age, and intestinal epithelial barrier function (n = 8 per condition) at 20 weeks of age. (I) Inflammatory cytokine RNA abundance. (J) H&E-stained colon sections at 32 weeks of age. Graphs are representative of 10 fields of view (n = 4 mice per condition). Scale bars, 100 μm. (D to G) Mice at 20 weeks of age. (A and C) n = 32 per condition. (B and I) n = 30 per condition. (D to F) n = 6 per condition. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05; one-way ANOVA with Tukey’s multiple comparisons test (A to J).

  • Fig. 8 Model of intestinal inflammation due to recurrent Gram-negative ST infection.

    In the absence of infection of the small intestine, the anti-inflammatory GPI-linked IAP glycoprotein (green circles) is highly expressed on the enterocyte cell surface. IAP is eventually released into the lumen and travels through the intestinal tract to the colon where it detoxifies LPS-phosphate produced by Gram-negative and commensal bacteria via dephosphorylation (yellow circles). Nascent IAP at the enterocyte cell surface undergoes a low rate of desialylation linked to the rate of internalization and degradation involving a normal mechanism of IAP aging and turnover. Enterocytes of the small intestine respond to LPS-phosphate and ST infection by activating TLR4 function, which induces host NEU3 Neu (blue bars) on the enterocyte surface. Increased Neu activity accelerates the rate of IAP desialylation and internalization (orange circles), reducing IAP abundance and resulting in increased levels of LPS-phosphate in the colon where TLR4 activation elicits inflammation and disease.

  • Recurrent infection progressively disables host protection against intestinal inflammation

    Won Ho Yang, Douglas M. Heithoff, Peter V. Aziz, Markus Sperandio, Victor Nizet, Michael J. Mahan, Jamey D. Marth

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