Japanin modulates dendritic cell maturation, rather than simply inhibiting it.

<p>Dendritic cells were cultured in the presence or absence of Japanin (500 ng/ml) and LPS (100 ng/ml) for 18–20 hours. (A) CD40, CD83, CD86, CD274 and HLA-DR expression were then assessed by flow cytometry, and (B)the concentration of pro-inflammatory cytokines in the culture supernatant was measured by Luminex1. Modelled means ±95% confidence intervals using data from at least four experiments are shown, except where marked ‡ where above-scale readings in the LPS-only made it impossible to calculate meaningful confidence intervals; the graphs show the lowest possible mean value (taking an above-scale value to be equal to the maximum possible on-scale value). ** p<0.01, * p<0.05, p<0.1, NS p>0.05.</p

Pretreatment of dendritic cells with Japanin inhibits their upregulation of CD86 in response to LPS.

<p>Dendritic cells were incubated with japanin for 24 hours prior to the addition of LPS (100 ng/ml) for a further 18–20 hours. CD86 expression was then analysed by flow cytometry. (A) The results from a representative experiment using 500 ng/ml japanin. (B) Titration of Japanin concentration, showing a dose-dependent inhibition of CD86 upregulation. The range and mean of duplicate measurements from one representative experiment are shown. This experiment was performed four times, with dose-dependency demonstrated each time, but with EC₅₀ varying between donors.</p

Japanin homologues modulate DC maturation.

<p>(A) Three Japanin homologues were successfully expressed in Sf9 cells, as shown by Western blotting with an anti-His tag antibody. ∼10 ng protein was loaded per lane. (B) & (C) Dendritic cells were cultured in the presence or absence of Japanin or Japanin homologues (500 ng/ml) and LPS (100 ng/ml) for 18–20 hours. CD86 (B) and CD274 (C) were then assessed by flow cytometry. Modelled means ±95% confidence intervals using data from three (cells with LPS) or four (cells without LPS) experiments are shown. * p<0.05, as compared to cells without Japanin or a Japanin homologue.</p

Japanin blocks differentiation of DC from monocytes.

<p>Monocytes were cultured with GM-CSF (1000 U/ml) and IL4 (500 U/ml) with or without Japanin (500 ng/ml). Before the culture, and again after 3 and 5 days of culture, CD1a and CD14 expression were assessed by flow cytometry, in order to monitor differentiation into CD1a⁺CD14^low dendritic cells. Data shown is from one experiment, representative of three independent experiments using cells from different donors.</p

Japanin inhibits CD86 upregulation in response to multiple DC maturation stimuli.

<p>(A) Dendritic cells were cultured for 18–20 hours in the presence or absence of Japanin (500 ng/ml) and stimuli: (25 µg/ml Poly(I:C), via TLR3; 100 ng/ml LPS, TLR4; 4 µg/ml CL097, TLR7/8; 20 ng/ml IFNα2, IFNAR; 10–12.5 ng/ml TNFα, TNFR; 20 ng/ml IFNγ, IFNGR). CD86 expression was then assessed by flow cytometry. Modelled means ±95% confidence intervals using data from at least four experiments are shown, except for CL097 for which three experiments were performed, using cells from a total of five donors. (B) Data from all these experiments was used to assess the effect of Japanin on CD86 expression in the absence of stimuli. ** p<0.01, * p<0.05, NS p>0.05.</p

Japanin-like proteins form a clade within hard tick lipocalins.

<p>(A) A phylogenetic tree derived from maximum-likelihood analysis of hard tick lipocalins (including Japanin and its homologues), as well as the soft tick lipocalins OmCI, monomine and Am182. Sequences were aligned using ClustalX and manually refined, then Mega5.1 was used to construct a phylogeny. The frequency with which associated taxa clustered together in the bootstrap test is shown. For reasons of clarity, only selected protein names and bootstrap frequency labels are shown, and the Japanin clade is shown in detail in (B), with the full tree supplied as supplementary data.</p

Sequence alignment of Japanin and its homologues.

<p>Alignments were generated with ClustalX and manually refined. Shading intensity indicates BLOSUM62 score. N-glycosylation sequences and conserved cysteine residues are boxed. Ra-FS-HBP2 (PDB ID 1QFT) is aligned as an example of a tick lipocalin with low sequence similarity to Japanin.</p

Cross-linked gp120 sensitizes DC through DC-SIGN and MCLRs for CD40L-mediated apoptosis.

<p>(<b><i>A</i></b>, <b><i>B</i></b>) moDC were pretreated with anti-DC-SIGN mAbs or isotype control Ab before pulse with cross-linked gp120_ADA and co-culture for 3 d with autologous activated CD4 T cells, and subsequently subjected to cell viability assay. Data are representative of 3 experiments and are expressed as mean ± SD from 3 experiments in <b><i>B</i></b>. (<b><i>C, D</i></b>) moDC were treated with cross-linked recombinant gp120_ADA with or without pre-treatment by soluble ICAM-3-Fc chimeric protein, anti-CD4 plus anti-CCR5 mAbs, or anti-DC-SIGN mAbs. Cells were subsequently co-cultured with mock- or CD40L-transfected (CD40L Tf) cells for 3 d. Data are representative of 7 experiments in panel <b><i>C</i></b> and are expressed as mean ± SD (n = 7) in <b><i>D</i></b>; ***p<0.005. (<b><i>E, F</i></b>) Recombinant gp120_ADA were treated with or without EndoH, and then cross-linked with anti-His Ab before use to pulse moDC. Prior to gp120 pulsing, moDC were pre-treated with or without mannan or FcR blocking reagent, or anti-DC-SIGN mAbs for 30 minutes. After gp120 pulsing, DC were subsequently cocultured with CD40 Tf for 3 days. DC without any pre-treatment and only pulsed with anti-His Ab were used as a control (control DC). Data are representative of 3 experiments and expressed as mean ± SD from 3 experiments in <b><i>F</i></b>; *p<0.05, **p<0.01.</p

Sera from HIV-1(+) individuals can sensitize moDC for DC-SIGN dependent CD40L-mediated apoptosis.

<p>(<b><i>A</i></b>) moDC were treated with HIV(+) serum before or after immunoprecipitation (IP) with anti-gp120 mAbs, or with or without anti-DC-SIGN or isotype control mAbs, and subsequently co-cultured with autologous activated CD4 T cells. After 3 d, cells were harvested and subjected to TUNEL assays. Cell death was assessed as the percentage of cells expressing terminal deoxynucleotidyl transferase (TdT). DC pulsed with HIV(+) serum without coculture with activated CD4 T cells (top panel) were also used as a control. Data are representative of 4 experiments. (<b><i>B</i></b>) MoDCs were treated with anti-DC-SIGN mAbs, isotype control Ab, or anti-CD40L mAb before pulse with HIV serum (before or after immunoprecipitation of gp120) and cocultured with activated CD4 T cells. Data are expressed as mean ± SD (n = 4); *p<0.05 and **p<0.01 compared with ‘HIV(+) serum-DC plus isotype Ab’. (<b><i>C</i></b>,<b><i>D</i></b>) moDC were treated with normal AB serum or HIV-1(+) serum with viral RNA copies >400,000/ml (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003100#ppat.1003100.s013" target="_blank">Table S1</a>), with or without pre-treatment with anti-CD4 plus chemokine receptor or anti-DC-SIGN mAbs, or with gp120-depleted (immunoprecipitated, IP) HIV(+) serum, and co-cultured with CD40L Tf for 3 d. Data are representative of 4 experiments in panel <b><i>C</i></b> and individual datum with the mean is shown in panel <b><i>D</i></b>; †: P<0.001 between with and without IP of gp120 from the HIV serum, **: P<0.01 between with and without pre-treatment with anti-DC-SIGN mAbs.</p

Cross-linked recombinant gp120 sensitizes moDC for CD40L-mediated apoptosis after co-culture with activated CD4 T cells.

<p>(<b><i>A</i></b>) moDC were treated for 24 h with anti-His mAb alone (control DC, left panels) or with 25 nM gp120_ADA cross-linked with anti-His mAb (gp120-DC, right panels), and co-cultured with autologous activated (upper panels) or naïve (lower panels) CD4 T cells for 3 d. The moDC (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003100#ppat.1003100.s001" target="_blank">Fig. S1</a>) were analyzed for Annexin V (AV) and propidium iodide (PI) expression to assess the extent of apoptosis, as manifested by the percentage of the AV-positive [AV(+)] cells. Data are representative of 5 experiments. (<b><i>B</i></b>) Apoptosis of moDC was analyzed after treatment with different concentrations of cross-linked recombinant gp120_ADA or gp120_HXBc2 and co-culture with activated CD4 T cells for 3 d. DC treated with monomeric gp120 (not cross-linked with anti-His or anti-FLAG mAb) were used as a control. Data represent mean ± SD from 5 experiments; **p<0.01. The use of cross-linked recombinant gp120_BAL gave similar results (not shown). (<b><i>C</i></b>) Apoptosis of moDC was analyzed after treatment with the indicated cross-linked recombinant gp120, or appropriate mAb controls, at the indicated time points after co-culture with activated CD4 T cells. Data represent mean ± SD from 5 experiments (data for anti-His and anti-FLAG controls were indistinguishable); *P<0.05 and **P<0.01 compared with control group (DC with no gp120 pulse or DC plus anti-His/FLAG Ab). (<b><i>D</i></b>) moDC were respectively not treated (Control DC), or treated with gp120_ADA cross-linked with mouse IgG2a anti-His mAb (dimeric gp120-DC), or treated with cross-linked gp120_ADA supplemented with isotype control mouse IgG (gp120-DC+isotype IgG), and subsequently co-cultured with autologous activated CD4 T cells for 3 days before AV/PI staining. Data are representative of 3 experiments. (<b><i>E</i></b>) moDC were treated with cross-linked gp120_ADA and co-cultured for 3 d with autologous activated or naïve CD4 T cells, that had been pre-treated without or with 10 µg/ml isotype control or anti-CD40L mAb, before cell viability analysis. Data are expressed as mean ± SD from 5 experiments. **p<0.01.</p