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

Anti-CXCR2 antibody treatment increased susceptibility of non-antibiotic treated mice to <i>E</i>. <i>histolytica</i>.

<p>Rat anti-mouse CXCR2 monoclonal antibody was administered 2 hours prior to <i>E</i>. <i>histolytica</i> challenge to blockade CXCR2. <b>(a, b)</b> Whole cecal tissue or 100 μL peripheral blood was collected 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 shown as cell number in whole cecum (data are representative of similarly conducted two independent experiments, n = 3 per each group). <b>(c)</b> Myeloperoxidase activity was assessed by lysing 50 mg of cecum in hexadecyltrimethylammonium bromide buffer. Enzyme activity was calculated from standards using recombinant proteins and is shown normalized to total protein concentration. <b>(d)</b> Parasite burden in cecal content was measured by qPCR using 200 μL of cecal content. <b>(e)</b> <i>E</i>. <i>histolytica</i> induced epithelial barrier disruption was assessed by immunohistochemistry staining of cecal tissue targeting <i>E</i>. <i>histolytica</i> derived secreted protein, <i>E</i>. <i>histolytica</i> migration inhibitory factor (EhMIF) (data are presented as mean value of histology score blindly scored by three independent observers). <b>(f)</b> <i>Ex vivo</i> culture positivity was also assessed by placing 200 μL of cecal content in TYI media. *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i> by one-way ANOVA-test (a-e) or chi-squared test (d). NS, not significant. Error bars represent s.e.m. MPO, myeloperoxidase; EhMIF, <i>E</i>. <i>histolytica</i> migration inhibitory factor.</p

Antibiotic treatment promoted <i>E</i>. <i>histolytica</i> invasion with disrupted mucosal barrier.

<p>Parasite invasion and mucosal barrier disruption upon <i>E</i>. <i>histolytica</i> challenge were assessed using cecal tissue or cecal content at 24 hours after challenge. <b>(a, b)</b> Parasite burden in cecal content was measured by qPCR and <i>ex vivo</i> culture positivity was also assessed by putting 200 μL of cecal content to TYI media (n = 8 per group). <b>(c-g)</b> <i>E</i>. <i>histolytica</i> induced epithelial barrier disruption was assessed by immunohistochemistry staining of cecal tissue targeting <i>E</i>. <i>histolytica</i> derived secreted protein, <i>E</i>. <i>histolytica</i> migration inhibitory factor (EhMIF) (representative picture in Fig 3c). Mucin containing goblet cells were stained by Periodic acid-Schiff and immunohistochemistry staining of cecal tissue targeting MUC2 (data are presented as representative pictures (e, f). Data are also presented as mean value of histology score blindly scored by three independent observers from similarly conducted two independent experiments, n = 8 per group (d, g)). <b>(h)</b> Cecal lysate at 24 hours after <i>E</i>. <i>histolytica</i> challenge was tested for Muc-2 gene expression by qRT-PCR and is shown normalized to the GAPDH housekeeping gene. <b>(i)</b> Cecal cytokine was assessed by lysing 50 mg of cecal sections and quantifying protein via ELISA, and shown normalized to total protein concentration (n = 5 per group (g, h)). <b>(j,k)</b> whole cecal tissue was isolated and processed to a single cell suspension and stained for flow cytometry. Eosinophils (CD45⁺ CD11b⁺ SiglecF⁺) were quantified and shown as ratio to CD45⁺ subsets or cell number in whole cecum (n = 8 per 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, h, i, & k), Mann-Whitney U-test (d & g) or chi-squared test (b). NS, not significant. Error bars represent s.e.m. Eh MIF, <i>E</i>. <i>histolytica</i> migration inhibitory factor.</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

Diversity of microbiome was decreased during amebic colitis.

<p>Diarrhea samples were from children in PROVIDE study (n = 20) and colonization samples were from children in NIH birth cohort (n = 47), both of which were followed longitudinally in an urban slum in Dhaka, Bangladesh. <b>(a)</b> The Shannon diversity index was examined using stool samples between 2 groups. <b>(b)</b> Age in days when stool samples were collected was also compared. *<i>P<0</i>.<i>05</i>, by Welch’s unequal variance t-test. Error bars represent standard error of the mean (s.e.m).</p

Antibiotic pre-treatment rendered C57BL/6 mice susceptible to <i>E</i>. <i>histolytica</i> colitis.

<p><b>(a)</b> The Shannon diversity index of stool samples collected from antibiotic treated mice (n = 10) was compared with those from untreated control (n = 10) (C57BL/6 mice which were not challenged with <i>E</i>. <i>histolytica</i>). Then, they were infected with 2 x 10⁶ <i>E</i>. <i>histolytica</i> trophozoites intracecally. <b>(b, c)</b> Body weight changes and clinical illness score were measured for assessing systemic disease severity. <b>(d)</b> Fecal occult blood was examined for assessing intestinal damage. <b>(e)</b> Parasite burden in stool was assessed by qPCR. <b>(f)</b> Lipocalin-2 (Neutrophil Gelatinase-Associated Lipocalin) in stool was measured for assessing neutrophil mediated gut inflammation. *<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, b, e& f), Mann-Whitney U-test (c) or chi-squared test (d). NS, not significant. Error bars represent s.e.m.</p