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

Fig 4

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

Neutrophil recruitment to the infection site and its activation upon E. histolytica challenge were assessed using cecal tissue at 24 hours after challenge. (a) 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-d) 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). (e-g) Whole cecal tissue was isolated and processed to a single cell suspension and stained for flow cytometry. Neutrophils (CD45+ CD11b+ Ly6Ghigh, representative gating is shown at Fig 3i) were quantified and shown as ratio to CD45+ subsets or cell number in whole cecum (n = 8 per each group). (i-j) 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). *P<0.05, **P<0.01, ***P<0.001 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.

Fig 4

doi: https://doi.org/10.1371/journal.ppat.1006513.g004