The p50 Subunit of NF-κB Orchestrates Dendritic Cell Lifespan and Activation of Adaptive Immunity

<div><p>Dendritic cells play a central role in keeping the balance between immunity and immune tolerance. A key factor in this equilibrium is the lifespan of DC, as its reduction restrains antigen availability leading to termination of immune responses. Here we show that lipopolysaccharide-driven DC maturation is paralleled by increased nuclear levels of p50 NF-κB, an event associated with DC apoptosis. Lack of p50 in murine DC promoted increased lifespan, enhanced level of maturation associated with increased expression of the proinflammatory cytokines IL-1, IL-18 and IFN-β, enhanced capacity of activating and expanding CD4⁺ and CD8⁺ T cells <em>in vivo</em> and decreased ability to induce differentiation of FoxP3⁺ regulatory T cells. In agreement, vaccination of melanoma-bearing mice with antigen-pulsed LPS-treated p50^−/− BM-DC boosted antitumor immunity and inhibition of tumor growth. We propose that nuclear accumulation of the p50 NF-κB subunit in DC, as occurring during lipopolysaccharide-driven maturation, is a homeostatic mechanism tuning the balance between uncontrolled activation of adaptive immunity and immune tolerance.</p> </div

Lack of p50 increases DC maturation.

<p>(A) Endocytic activity. Left: data represent mean ± SEM of 3 independent experiments performed in triplicate. Right: representative experiment showing the shift of wt BM-DC (dashed line) compared to p50^−/− BM-DC (solid line) (B and C) Cytofluorimetric analysis of MHC class I and II expression by wt and p50^−/− BM-DC stimulated with LPS for the indicated time. Data represent mean ± SEM (N = 4). (D) Cytofluorimetric analysis of MHC II expression by wt and p50^−/− splenic DC, 24 h after the injection of LPS (100 ng/g mouse). Left, data from 3 experiments (6 mice/group) are shown. Right, representative panel of MHC-II high and low CD11c⁺ cells. <b>*</b><i>P</i><0.05 <b>**</b><i>P</i><0.01, <i>t</i> test.</p

Lack of p50 increases DC survival.

<p>(A) Cytofluorimetric analysis of apoptotic BM-DC upon LPS stimulation. Data represent mean ± SEM (N = 7) (B) Cytofluorimetric analysis of splenic DC survival 24 h and 48 h after LPS injection in mice. Data represent mean ± SEM of 3 independent experiments (each dot corresponds to 1 mouse). (C) Analysis of MHC II⁺ splenic DC survival 24 h after the injection of 100 ng LPS/g (animal weight). <i>In situ</i> apoptosis of splenic DC was determined by TUNEL staining. Results are representative of 3 independent experiments (4 mice/group). (D) Bax protein levels in wt and p50^−/− BM-DC stimulated with LPS for the indicated time. Left panel, 1 of 2 independent experiments is shown. Right panel, mean ± SEM (N = 2). (E) <i>In vitro</i> BM-DC migration of wt and p50^−/− BM-DC. 24 h LPS stimulated BM-DC were seeded in a boyden chamber and migration towards CCL-19 was evaluated after 90′ incubation. (F) <i>In vivo</i> migration of wt and p50^−/− BM-DC to draining lymph nodes. BM-DC unstimulated or stimulated with LPS for 24 h were labeled with a fluorescent dye and injected into the footpads of wt mice. Popliteal draining lymph nodes were recovered 24 h and 48 h later and percentage of migrated BM-DC was evaluated by flow cytometry (N = 4−15). *<i>P</i><0.05 <b>**</b><i>P</i><0.01, <i>t</i> test.</p

Lack of p50 improves DC vaccination ability.

<p>(A) Vaccination of wt recipient mice with wt or p50^−/− BM-DC loaded with either OVA or an irrelevant protein. Vaccinated mice were injected with B16-OVA melanoma cells and tumor growth evaluated at different times. Data from one of two independent experiments (6 mice/group) are shown. (B) <i>Ex vivo</i> IFN-γ secretion by splenocytes recovered at day 30 after vaccination. Data represent the mean ± SEM of 6 mice/group. (C, D) Human mo-DC were transfected with a p50-specific Stealth RNAi siRNA (p50 siRNA) or with a Stealth negative control (scramble), incubated with or without LPS (100 ng/ml) for 48 h. Next cells were washed and lysed to analyzed p50 levels by western blotting (C) or added to purified allogeneic T cells (ratio mo-DC:T cells 1∶10) (D). After 5 days, supernatants were collected and tested by ELISA. Data represent mean ± standard deviation of 1 of 3 independent experiments performed in triplicate. <b>*</b><i>P</i><0.05, <b>**</b><i>P</i><0.01, <i>t</i> test.</p

p50 NF-κB regulates the antigen-presenting capacity of DC.

<p>(A) Kinetic of nuclear accumulation of p50 NF-κB in wt BM-DC stimulated with LPS. One of 3 independent experiments with similar results is shown. (B) IFN-γ secretion by CD4+ T cell co-cultured with wt or p50^−/− untreated or LPS-treated BM-DC loaded with a MHC II-restricted peptide. Data represent mean ± SEM (N = 5). (C-E) wt or p50^−/− untreated or LPS-treated BM-DC loaded with a MHC II-restricted peptide were injected in the footpad of wt recipient mice. Next, <i>ex</i> vivo IFN-γ secretion by CD4+T cells from draining lymph nodes (C) and spleen (E) was measured. (F-I) wt or p50^−/− untreated or LPS-treated BM-DC loaded with a MHC I-restricted peptide were injected in the footpad of wt recipient mice. Next, <i>ex</i> vivo IFN-γ secretion by CD8+T cells from draining lymph nodes (F) and spleen (H) was measured. Lymph nodes and spleen were also analyzed for the presence of double positive IFNγ and CD4 (D) or CD8 cells (G) or OVA-specific CD8+ T cells (I). (L) wt or p50^−/− BM-DC were incubated with DQ ovalbumin at 37°C or 0°C and antigen processing was analyzed by mean of fold increase of M.F.I. compared to cells at time zero (dotted line: 0°C, straight line: 37°C). Data represent mean ± SEM (N = 2; 6 mice/group). <b>*</b><i>P</i><0.05 <b>**</b><i>P</i><0.01, <i>t</i> test.</p

Lack of p50 impairs the immunoregulatory activity of DC.

<p>(A) <i>In vitro</i> generation of Foxp3⁺ T cells by wt and p50^−/− BM-DC. Data represent mean ± SEM (N = 3). (B) Cytofluorimetric analysis of naturally occurring Foxp3⁺ T cells in spleens and lymph nodes of wt and p50^−/− mice. Histograms show the % of Foxp3⁺ cells among CD4⁺ cells. (C) Effect of 1-methyl DL-tryptophan (1 MT; 10 µM; Sigma Aldrich) on the secretion of IFN-γ by OVA specific CD4⁺ T cell, in response to BM-DC loaded with the CD4⁺ T cell specific OVA_323–339 peptide and treated or not for 24 h with 100 ng/ml LPS and 200 U/ml IFN-γ. Data represent 1 of 3 independent experiments. <b>*</b><i>P</i><0.05, <i>t</i> test.</p

Lack of p50 in DC favors a Th1-promoting cytokine profile.

<p>(A) IL-12p70, IL-18 and IFN-β secretion by wt and p50^−/− BM-DC stimulated with LPS or LPS and IFN-γ for 24 h. Data represent mean ± SEM (N = 3). IL-10, TNFα and IL-1β secretion by wt and p50^−/− BM-derived (B) and splenic DC (C) stimulated with LPS for 24 h. Data represent mean ± SEM (N = 5 and 3, respectively). <b>*</b><i>P</i><0.05 <b>**</b><i>P</i><0.01, <i>t</i> test.</p

IL-1-mediated transcriptional regulation of β-glucan-induced late immunoregulatory cytokines in human DCs.

<p>(A) Human monocyte-derived DCs were cultured with or without particulate β-glucan and IL-1RA (2.5 µg/ml). Transcript accumulation was determined by absolute Real-Time qRT-PCR using total RNA and primers (Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114516#pone.0114516.s001" target="_blank">File S1</a>) for the detection of either primary or mature transcripts. Results are mean, n = 4. The times when significant differences were found as well as their p values are indicated in the figure. (B) Human monocyte-derived DCs were cultured in the presence or absence of particulate β-glucan and IL-1RA (25 µg/ml) for 6 h. ChIP was performed using sheared chromatin from fixed cells. DNA-protein complexes were immunoprecipitated with control IgG or RNAPII antibody (anti-RNAPII). Eluted DNA was analyzed by Real-Time qRT-PCR using primers (Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114516#pone.0114516.s001" target="_blank">File S1</a>) specific for <i>IL6</i>, <i>IL12B</i>, and <i>IL23A</i> promoter regions, proximal to the transcription start site. Results are mean ± SEM, n = 4. Statistics: paired t-test.</p

Regulation of the DC response to β-glucan by the IL-1-induced nuclear factor IκB-ζ.

<p>(A) Human monocyte-derived DCs were cultured with or without particulate β-glucan and/or IL-1RA (25 µg/ml). Gene expression data are from the experiment of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114516#pone-0114516-g004" target="_blank">Figure 4A</a> and results shown are normalized mRNA counts from technical triplicates from one representative donor out of 8 analyzed. (B) Human monocyte-derived DCs were cultured for 12 h in the indicated condition. IL-1RA was used at 250 µg/ml. Total extracts were analyzed by Western blotting with the indicated antibodies. Data shown are from one representative donor out of 9 tested. Arrows indicate specific bands, while asterisks show nonspecific bands. (C) Human monocyte-derived DCs were cultured in the presence or absence of particulate β-glucan and/or IL-1RA as indicated for 6 h and nuclear extracts prepared for Western blotting. Data are from one donor and are representative of those obtained in at least 3 independent experiments with cells from different donors. Arrows indicate specific bands, while asterisks nonspecific bands. (D) Human monocyte-derived DCs from different donors were stimulated for 12 h with β-glucan in presence of transfection reagent (none) after pre-incubation with a non-targeting siRNA pool (ctr siRNA) or a pool of four siRNA directed against <i>NFKBIZ</i> (<i>NFBIZ</i> siRNA). mRNA levels in cell lysates for 64 selected genes were quantitated by NanoString's nCounter technology and the map shows genes with significant changes in expression (FDR<0.1) following knockdown of <i>NFKBIZ</i>. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114516#pone-0114516-g003" target="_blank">Figure 3</a>.</p

IFN-γ-mediated regulation of immune response to β-glucan <i>via</i> inhibition of IL-1β and IκB-ζ in activated DCs.

<p>(A) Human monocyte-derived DCs, unprimed or primed overnight with IFN-γ were stimulated or not for 12 h with β-glucan as indicated. mRNA levels in cell lysates for 64 selected genes were quantitated by NanoString's nCounter technology and the map shows genes with significant changes in expression (FDR<0.1) upon IFN-γ. Results from 3 different donors are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114516#pone-0114516-g003" target="_blank">Figure 3</a>. (B) Human monocyte-derived DCs, unprimed or primed overnight with IFN-γ were stimulated or not for 24 h with β-glucan as indicated. Protein secretion in the culture supernatants was measured by ELISA. Results are mean ± SEM, n = 7. Statistics: Tobit model analysis. (C) Human monocyte-derived DCs, unprimed or primed overnight with IFN-γ were cultured in the presence or not of particulate β-glucan as indicated for 6 h and nuclear extracts prepared for Western blotting. Data are from one donor and are representative of those obtained in at least 3 independent experiments with cells derived from different donors. Arrows indicate specific bands, while asterisks nonspecific bands. (D) Optical density of nuclear IκB-ζ bands from Western blotting analysis in (C). Data shown are fold induction over background from unstimulated cells. Results are mean ± SEM, n = 3. Statistics: t-test.</p