Interleukin-25 Mediated Induction of Angiogenin-4 Is Interleukin-13 Dependent

<div><p>The intestinal surface is directly exposed to both commensal microorganisms as well as pathogens with a single layer of epithelium separating luminal microorganisms from internal tissues. Antimicrobial peptides play a crucial role in allowing epithelial cells to contain in the lumen beneficial and pathogenic microorganisms. The commensal dependent, epithelial produced, Th2 cytokine IL-25 can induce IL-13 and potentially the antimicrobial peptide angiogenin-4. Here we show that IL-13 downstream of IL-25 is required to induce angiogenin-4. IL-25 mediated induction of angiogenin-4 is furthermore not dependent on IL-22 or IL-17.</p></div

Depletion of IL-13 abrogates rIL-25 induction of angiogenin-4.

<p>CBA/J mice were treated with 0.5 micrograms of recombinant IL-25 each day for a total 6 doses. Control mice received PBS. Recombinant IL-25 treated mice received 200μg anti-IL-13 antibody or isotype control on day 3 and on day 5. Angiogenin-4 relative expression was measured from cecal tissue, n = 5–8 (A). Histological scoring (1 to 5; low to high) for Angiogenin-4 in cecum from mice treated with PBS or rIL-25 with isotype control or rIL-25 with anti-IL-13 (B). Representative IHC staining for angiogenin-4 in samples of cecum tissue from PBS treated (C) or rIL-25 with isotype control (D) or rIL-25 with anti-IL-13 treated mice (E).</p

rIL-25 administration induces IL-13 expression.

<p>CBA/J mice were treated with 0.5 micrograms of recombinant IL-25 (closed square, n = 8) each day for a total of 5 doses and control mice received PBS (open circle, n = 7). IL-13 relative expression was measured from mouse cecal tissue and normalized with house keeping gene GAPDH and β actin.</p

rIL-25 administration induces angiogenin-4 expression.

<p>CBA/J mice were treated with 0.5 micrograms of recombinant IL-25 (triangle, n = 11) each day for a total of 10 doses over 10 days. Control mice received PBS (open circle, n = 11). Angiogenin-4 relative expression was measured from mouse cecal tissue and normalized with house keeping gene GAPDH and β actin (A). Histological scoring (1 to 5; low to high) for Angiogenin-4 in cecum from mice treated with PBS or rIL-25 (B). Representative IHC staining for angiogenin-4 in samples of cecum tissue from PBS treated (C) or rIL-25 treated mice (D).</p

rIL-13 administration induces angiogenin-4 expression.

<p>CBA/J mice were treated with 0.5 micrograms of recombinant IL-13 (triangle, n = 5) on each day for total 4 doses. Control mice received PBS (open circle, n = 5). Angiogenin-4 relative expression was measured from mouse cecal tissue and normalized with house keeping gene GAPDH and β actin (A). Histological scoring (1 to 5; low to high) for Angiogenin-4 in mouse cecum from PBS or rIL-13 treated mice (B). Representative IHC staining for angiogenin-4 in samples of cecum tissue from PBS treated (C) or rIL-13 treated mice (D).</p

rIL-25 administration increases angiogenin-4 expression in a dose dependent manner.

<p>CBA/J mice were treated with 0.5 micrograms of rIL-25 each day for a total 4 doses (triangle, n = 7) or 8 doses (inverse triangle, n = 5) and control mice received 4 or 8 doses of PBS (open circle, n = 7 for 4 doses and n = 5 for 8 doses). Angiogenin-4 relative expression was measured from mouse cecal tissue and normalized with house keeping gene GAPDH and β actin.</p

rIL-25 induced angiogenin-4 production is not significantly influenced by IL-17 or IL-22 blockade.

<p>CBA/J mice were treated with 0.5 micrograms of recombinant IL-25 each day for total 7 doses and control mice received PBS. rIL-25 treated mice received 200μg anti IL-17 antibody (inversed triangle) or 200μg anti IL-22 antibody (open square) or 200μg anti-IL-13 antibody (open circle), or isotype control on day 3, on day 5 and on day 7. Angiogenin-4 relative expression was measured from cecal tissue.</p

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis

<div><p>The disease severity of <i>Entamoeba histolytica</i> infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of <i>E</i>. <i>histolytica</i> virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic <i>E</i>. <i>histolytica</i> infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to <i>E</i>. <i>histiolytica</i> infection) were treated with antibiotics prior to cecal challenge with <i>E</i>. <i>histolytica</i>. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of <i>E</i>. <i>histolytica</i>. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1β, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.</p></div

Graphical summary: Effects of microbiota on immune response to <i>E</i>. <i>histolytica</i> infection.

<p>During dysbiostic state (bottom figures), diversity of gut commensal microbes is low compared to healthy state (upper figures). IL-25 mediated mucosal protection by epithelial cells (1) and CXCR2 expression on neutrophils (2) upon <i>E</i>. <i>histolytica</i> challenge are diminished in dysbiostic state, allowing more severe tissue invasion by <i>E</i>. <i>histolytica</i>.</p

Neutrophil activation and recruitment and CXCR2 expression were decreased in antibiotic pre-treated mice.

<p>Neutrophil recruitment to the infection site and its activation upon <i>E</i>. <i>histolytica</i> challenge were assessed using cecal tissue at 24 hours after challenge. <b>(a)</b> Myeloperoxidase activity was assessed by lysing 50 mg cecal lysate in hexadecyltrimethylammonium bromide buffer. Enzyme activity was calculated from standards using recombinant proteins and is shown normalized to total protein concentration (n = 3 or 5 per group). <b>(b-d)</b> Cecal cytokines were assessed by lysing 50 mg of cecal sections and quantifying protein via ELISA, and shown normalized to total protein concentration (n = 3 or 5 per group). <b>(e-g)</b> Whole cecal tissue was isolated and processed to a single cell suspension and stained for flow cytometry. Neutrophils (CD45⁺ CD11b⁺ Ly6G^high, representative gating is shown at <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006513#ppat.1006513.g003" target="_blank">Fig 3i</a>) were quantified and shown as ratio to CD45⁺ subsets or cell number in whole cecum (n = 8 per each group). <b>(i-j)</b> Localization of neutrophils was assessed by immunohistochemistry staining of cecal tissue targeting Ly6G. Mucosal thickness (orange line) was calculated as mean vale of the data from 6 different portions (i), cell number was calculated as mean vale of the data from 3 different portions (data are presented as representative picture (h), or data from n = 8 per each group). *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i> by Welch’s unequal variance t-test (a-g, i and k), Mann-Whitney U-test (j) or chi-squared test (b). NS, not significant. Error bars represent s.e.m.</p