MDDC TLR-2 and DC-SIGN are required for appropriate cytokine responses to <i>Lb</i>. <i>rhamnosus</i> JB-1.

<p><i>(A) Lb</i>. <i>rhamnosus</i> JB-1 or <i>Lb</i>. <i>murinus</i>-stimulated MDDCs were pre-incubated with blocking antibodies for TLR-2 (n = 8 donors), DC-SIGN (n = 8 donors) or antibody isotype controls for 30 minutes prior to stimulation and cytokine secretion was analyzed after 24 hours. Results are expressed as the mean pg/ml +/− standard error. (B) TLR-2 activation was confirmed using HEK-293 cells which express TLR-2. *p<0.05</p

<i>Lb</i>. <i>rhamnosus</i> JB-1 is bound by MDDC DC-SIGN.

<p>(A) <i>Lb</i>. <i>rhamnosus</i> JB-1 binding to MDDCs is partially dependent on the presence of Ca²⁺ ions, suggesting the involvement of CLRs. The flow cytometry plots are representative of 3 independent experiments. (B) As visualized by confocal microscopy, blockade of DC-SIGN decreased <i>Lb</i>. <i>rhamnosus</i> JB-1 binding to MDDC, while neutralizing TLR-2 antibodies had no effect on <i>Lb</i>. <i>rhamnosus</i> JB-1 binding.</p

MDDC costimulatory molecule expression and cytokine secretion.

<p>(A) CD80 and CD86 were upregulated on MDDCs after exposure to both bacterial strains. LPS served as a positive control. Co-stimulatory molecule expression was significantly higher after stimulation with <i>Lb</i>. <i>murinus</i> compared with <i>Lb</i>. <i>rhamnosus</i> JB-1. (B) <i>Lb</i>. <i>murinus</i> stimulated MDDC cytokine secretion was significantly higher, for all cytokines tested, compared to that induced by <i>Lb</i>. <i>rhamnosus</i> JB-1 (n = 5 donors). *p<0.05</p

<i>Lb</i>. <i>rhamnosus</i> JB-1 internalisation by MDDCs.

<p>(A) Following antibiotic treatment, which kills bacteria outside MDDCs, the peak recovery of viable <i>Lb</i>. <i>rhamnosus</i> JB-1 was observed after 48 hours, suggesting that this was the time point when the bacteria were internalized by the MDDCs. (B) MDDC phagocytosis of another bacterial strain, <i>E</i>. <i>coli</i>, was reduced when the MDDCs were co-exposed to <i>Lb</i>. <i>rhamnosus</i> JB-1, but not <i>Lb</i>. <i>murinus</i>.</p

Differential MDDC binding and processing of two <i>Lactobacilli</i> strains.

<p>MDDCs bind <i>Lb</i>. <i>rhamnosus</i> JB-1 efficiently but intracellular processing is delayed, as visualized by multispectral flow cytometry imaging (A) and confocal microscopy (B). <i>Lb</i>. <i>murinus</i> is also efficiently bound by MDDCs and is rapidly processed (C). For both strains, bacterial cells are CFSE labeled (green), while CD11c⁺ cells are PE-Cy5 stained (red).</p

B. longum 35624 EPS proton and carbon signals.

<p>(<b>A</b>) A selected region of the multiplicity-edited, gradient enhanced ¹H, ¹³C-HSQC NMR spectrum of the exopolysaccharide. Letters denote the residues as given in the structural formula and arabic numerals denote the respective pyranose position. Resonances from anomeric carbons/protons, glycosylation sites and resolved signals are annotated. (<b>B</b>) Selected region of the ¹H, ¹³C-HSQC-TOCSY NMR spectrum (600 MHz) of the acid-treated <b>35624</b> EPS. Arabic numerals before and after oblique stroke denote carbons and protons, respectively.</p

B. longum 35624 EPS characterization.

<p>(A) The 600 MHz ¹H NMR proton spectrum of the acid-treated <b>35624</b> EPS (D₂O, 338 K) is illustrated. A part of the high-field region is displayed in the insert. <b>(B)</b> Expansion plot of the 150 MHz 13C NMR spectrum of the acid-treated <b>35624</b> exopolysaccharide. The anomeric signals on the left confirmed the presence of a hexasaccharide repeat unit.</p

Mild acid hydrolysis of EPS.

<p>(A) Separation of EPS fragments by PGC HPLC with MS/MS detection. The extracted ion chromatogram for mass 1008.39 Da shows four peaks. Their reducing end sugar was clearly revealed by ESI-MS/MS. Their assignment as either Gal or Glc and the interpretation in terms of fragment structures was done <i>a posteriori</i> based on MALDI-TOF data and on knowledge of the EPS structure. (B) Example of a MALDI-TOF/TOF fragment spectrum showing b-ions from the non-reducing and y- and y´ (= ^1,5x) -ions from the reducing end.</p

<i>Bifidobacterium longum</i> general genome features.

<p><i>Bifidobacterium longum</i> general genome features.</p

¹H and ¹³C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.

<p>¹H and ¹³C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.</p