Both sialylated and non-sialylated <i>C. jejuni</i> are scavenged by macrophages in the splenic marginal zone.

<p>Mice were injected with CFSE-labelled GB2 wt (A and C) or Cst-II mutant (B) bacteria and spleens were isolated after 1 h. Spleen sections were stained for sialoadhesin (A and B) or CD209b (C and D) and counterstained for B220 (purple). Insets show colocalisation of GB2 wt and ko bacteria with sialoadhesin and CD209b positive macrophages.</p

Increased type I interferon production in the spleens of GB2 wildtype injected animals.

<p>Mice were injected with PBS, GB2 wildtype or Cst-II mutant bacteria and spleens were isolated after 1 h. Spleen sections were homogenised for RNA extraction and cytokine expression was analysed by real-time PCR. Data is expressed as fold increase (mean ± sd; n = 4) compared to PBS-injected control animals. * p<0.05, Mann-Whitney U test.</p

Increased phagocytosis of sialylated <i>C. jejuni</i> by BM-MΦ.

<p>(A) BM-MΦ or (C) BM-DC were incubated with CFSE-labelled wt or Cst-II mutant GB11 for 60 min at 37°C (open histograms) or 4°C (filled histograms). Phagocytosis of <i>C. jejuni</i> by F4/80+ cells or CD11c⁺ cells was quantified as fold increase in fluorescence intensity using background fluorescence of 4°C, NaN₃ cultured cells as a reference. Phagocytosis of sialylated wt GB11 and unsialylated GB11 for 0, 15, 30 and 60 min shows that sialylation increases <i>C. jejuni</i> phagocytosis by BM-MΦ (B) but not DM-DC (D). One representative experiment out of 3 is shown (B and D; means ± sd of triplicates). * p<0.05; t-test.</p

Sialylated GB11 LOS induces higher cytokine levels by myeloid cells.

<p>BM-MΦ (A, C and E) and BM-DC (B, D and F) were incubated overnight (for IL-6 and IL-10) or for 4 h (IFN-β) with purified LOS derived from GB11 <i>C. jejuni</i> or Cst-II mutant <i>C. jejuni</i> and cytokine production was measured by ELISA. Sialylated LOS induces more IL-10 (A and B), IL-6 (C and D) and IFN-β (E and F) than non-sialylated LOS. Graphs represent pooled data as mean ± standard error from 3 (IL-10 and IFN-β) or 4 (IL-6) independent experiments. * p<0.05; paired t-test, n.d.: not detectable.</p

Sialylation of <i>C. jejuni</i> increases <i>in vivo</i> phagocytosis by splenic DC but not macrophages.

<p>Mice were sacrificed 1 h or 3 h after i.v. injection of PBS or CFSE-labelled GB2 (wt or Cst-II mutant). Bacterial deposition in spleen sections, stained for sialoadhesin, CD209b and B220, was quantified as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034416#s4" target="_blank">Materials and Methods</a> section. The CFSE-positive area of digital images was similar between GB2 wt and Cst-II mutant injected animals (A). No differences were observed in the CFSE-positive area within MOMA1 (B) or CD209b-positive cells (C). For each mouse, 6 pictures from 2 spleen sections were used for analysis (n = 4). Using Facs-analysis, the number of CFSE-positive metallophilic macrophages (Sialoadhesin+), red pulp macrophages (F4/80+), DC (CD11c+) and neutrophils (Ly-6G/C+) was determined per spleen (n = 3; D). Shown are means ± sd. * p<0.05; t-test with Welch's corrections.</p