Characteristics of LPS.

a<p>All are rough-type LPSs.</p

LPS with acylation defects induce functional mouse and human dendritic cells.

<p>BMDC were incubated overnight with OVA and activated for 8 h with different LPS. Stimulated DC were co-cultured with T cells from OT-I and OT-II <i>Rag-2^−/−</i> mice (A). The proliferation of OT-I and OT-II T cells was assessed after 3 days of co-culture by CFSE decrease. For T cell activation assays, the expression of surface markers such as CD25 and CD62L was analyzed by flow cytometry. At least 3 independent experiments were performed and one representative is shown. (B) CFSE-labeled allogeneic naïve CD4⁺ T and CD8⁺ T cells were co-cultured with activated mDC for 7 days. Cell division was tested by measuring CFSE-dilution Experiments were performed on 4 different donors. Data for one representative experiment are shown.</p

Kinetics of synthesis of pro-inflammatory cytokines.

<p>(A) BMDC were stimulated for 2 h, 4 h, 8 h or 24 h with medium (grey), <i>E. coli</i> hexa-acyl LPS (dark blue), <i>E. coli</i> tetra-acyl LPS (purple) or <i>Y. pestis</i> tetra-acyl LPS (light blue). Total RNA was purified from cell lysates, reverse transcribed and the amount determined by real-time quantitative PCR. Primers were used for qPCR amplification of <i>actin</i> (control), <i>p35, p40</i> and <i>TNF-α</i> genes. 3 independent experiments were done and one representative is shown, **p<0.01. (B) The secretion levels of IL-12p70, IL-12p40 and TNF-α were determined by ELISA. Data represent means ± standard errors of at least 4 independent experiments, **p<0.01. (C, D) BMDC were treated for 2 h and 4 h with medium, <i>E. coli</i> LPS (either hexa-acyl or tetra-acyl LPS) and <i>Y. pestis</i> tetra-acyl LPS. The intracellular synthesis of IL-12 (p40+p70) in (C) and TNF-α in (D) was analysed by flow cytometry. (E) The intracellular IL-12 and TNF-α production was studied in BMDC activated for 8 h with LPS variants. At least 3 independent experiments were performed and one representative is shown.</p

LPS with acylation defects induce semi-mature mouse and human dendritic cells.

<p>Mouse BMDC were stimulated for 8 h (in grey) and 24 h (in black) with medium, <i>E. coli</i> LPS (either hexa-acyl, penta-acyl or tetra-acyl) and <i>Y. pestis</i> tetra-acyl LPS. All LPS were used at the concentration of 100 ng/ml. MHC II and co-stimulatory molecules up-regulation on the cell surface was measured by flow cytometry (A) and cytokine secretion was determined by ELISA (B). Data represent means ± standard errors of at least 5 independent experiments, **p<0.01, *p = 0.01 to 0.05. Human blood mDC were stimulated overnight with medium (in grey), hexa-acyl <i>E. coli</i> LPS (in red), tetra-acyl <i>E. coli</i> LPS (in blue) and <i>Y. pestis</i> tetra-acyl LPS (in orange). Surface expression of HLA-DR, CD83, CD40 and CD86 was analyzed by flow cytometry (C) and cytokine levels in the culture supernatants were measured by Luminex (D). Experiments were performed on 4 different donors. The data for one representative are shown. ***p<0.001, **p<0.01, *p = 0.01 to 0.05.</p

Tetra-acyl LPS induce the activation of TLR4-dependent molecular pathways involved in mouse DC maturation.

<p>BMDC were activated with medium (grey), <i>E. coli</i> hexa-acyl LPS (dark blue), <i>E. coli</i> tetra-acyl LPS (purple) or <i>Y. pestis</i> tetra-acyl LPS (light blue) for 15 min, 30 min, 1 h and 2 h. NF-κB translocation was analyzed by confocal microscopy(A). Cells were fixed and stained for CD11c (in blue), MHC-II (in green) and NF-κB subunit p65/RelA (in red). The percentage of BMDC with translocated NF-κB into the nucleus was quantified (B). BMDC were stimulated for 30 min, 1 h, 4 h and 6 h with medium or different LPS. Cell lysates were subjected to SDS-PAGE and, after transfer to nitrocellulose, the membrane was probed with the antibodies against phospho-S6 (Ser235/236), S6 and an anti-actin antibody (C). Data represent means ± standard errors of at least 4 independent experiments, **p<0.01.</p

Tetra-acyl LPS induce effector molecules synthesis by human T cells.

<p>Human blood mDC were activated overnight either by medium or LPS variants and co-cultured with allogeneic naïve CD4⁺ T and CD8⁺ T cells. After 7 days, cells were incubated 6 h with PMA/Ionomycine in the presence of Brefeldin A. The intracellular levels of IFN-γ, IL-13 and IL-17 in CD4⁺ T (A) and IFN-γ and Granzyme B in CD8⁺ T cells (B) were analysed by flow cytometry. Experiments were performed on 4 different donors. Data for one representative experiment are shown.</p

Tetra-acyl LPS induce a degradation of IL-12 by the proteasome machinery in DC.

<p>BMDC were activated for 8 h with LPS variants in the presence or the absence of proteasome inhibitors such as epoxomycine (A) and Mg132 (B). The intracellular IL-12 (p40+p70) synthesis was then analysed. At least 3 independent experiments were performed and one representative is shown. (C) BMDC were activated for 2 h, 4 h, 8 h and 24 h with LPS variants and labelled with anti-MHC II(green), anti-CD11c (blue) and FK2 (red) antibodies to detect DALIS (white arrows). Quantification of the percentage of DC with DALIS at 2 h, 4 h and 8 h post-incubation with medium or post-stimulation with the different LPS. Quantifications were done by counting at least 300 cells in 3 independent experiments. Data represent means ± standard errors of at least 3 independent experiments, *p = 0.01 to 0.05.</p

Phospho-flow analysis of human IL-4 DC stimulated by LPS.

<p>Human IL-4 DC were activated by different LPS for 2 min, 5 min, 10 min, 30 min, 60 min and 180 min. A phospho-flow analysis using fluorescent cell barcoading was performed in order to assess the phosphorylation levels of molecules involved in TLR4 signaling. The heatmap visualization of phosphorylation changes is shown. The median fluorescent intensity (MFI) of stimulated cells is normalized by MFI of medium stimulated cells. Colored bar on the right shows the levels of fold changes. Experiments were performed on 4 different donors. The data for one representative are shown.</p