Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis

<div><p>Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR^CD11c-cre) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR^CD11c-cre mice, CD8⁺ DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c⁺ cells rescued LPS-induced loss of CD8⁺ DCs but not other DC subsets. Unlike wild-type animals, exposure of GR^CD11c-cre mice to low-dose LPS did not induce CD8⁺ DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.</p></div

Loss of endotoxin tolerance in GR^CD11c-cre mice.

<p>(A) Tolerizing dose of LPS-induced loss of CD8⁺ DCs in WT but not GR^CD11c-cre mice. Mice were treated with 0.5 μg/g body weight of LPS for 24 hr and rechallenged with 3 μg/g body weight of LPS for 3 hr before splenic DCs were analyzed by flow cytometry. The experiment was repeated three times, with three mice in each group per experiment. (B) High-dose LPS induced IL-12 production by DCs in GR^CD11c-cre mice despite prior exposure to low-dose LPS. Ten to twelve wk female GR^CD11c-cre mice (<i>n</i> = 4) and control mice (<i>n</i> = 5) were treated with 0.5 μg/g body weight of LPS for 24 hr and rechallenged with 10 μg/g body weight of LPS for 6 hr. Percentage of IL-12⁺ DCs in the spleen of a representative mouse is shown (left panel). Average numbers of total IL-12⁺ DCs were calculated based on the total number of splenocytes in each mouse (right panel). (C) Body temperature of mice rechallenged with 10 μg/g body weight of LPS 24 hr after tolerization. Results were pooled from two independent experiments, <i>n</i> = 8 for WT and <i>n</i> = 5 for GR^CD11c-cre. (D) Serum levels of TNF-α, IL-1β, IFN-α, and IFN-γ from mice in (B) were measured 6 hr after rechallenge. (E) Body temperature of mice rechallenged with 10 μg/g body weight LPS 24 hr after tolerization and 1 hr after injection with 250 μg anti-IL-12 or isotype control. Results were pooled from two independent experiments with one to two mice per condition, respectively, <i>n</i> = 3 per condition. The asterisk indicates the time by which all GR^CD11c-cre mice had died. All WT and the GR^CD11c-cre animals treated with anti-IL-12 survived. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

Elevated proinflammatory cytokines in GR^CD11c-cre mice.

<p>(A–C, E, and F) Kinetics of LPS-induced cytokine levels in WT and GR^CD11c-cre mice. The sera used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.g001" target="_blank">Fig 1C</a> were also used to determine cytokine concentrations at each time point. Data shown are from four to eight animals per strain per time point pooled from multiple independent experiments. (D) Semiquantitative RT-PCR analysis of IFN-α4 transcription in DC subsets from WT and GR^CD11c-cre mice 1 hr after LPS challenge. β-actin was used as internal control. Results shown are one representative of two independent experiments with pooled splenocytes from two WT and two GR^CD11c-cre mice. (G) Splenocytes were prepared from mice 12 hr after LPS injection, and cultured in vitro for 4 hr with Brefeldin A. Intracellular IFN-γ was measured for gated NK and T cells by flow cytometry. One mouse from each genotype is shown out of two independent experiments, each using three mice per group. (H) Mean fluorescence intensity (MFI) and percentage of IFN-γ⁺ cells in WT and GR^CD11c-cre mice 12 hr post LPS challenge (<i>n</i> = 3). (I) GR^CD11c-cre or WT mice, each represented by an individual symbol, were injected with the indicated doses of neutralizing anti-IL-12 antibody 1 hr before challenge with a lethal dose of LPS (10 μg/g body weight). Surface body temperature at 30 hr is plotted. Results shown are pooled from two independent experiments, each with three to four mice per group. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

GC suppresses in vivo IL-12 production by CD8⁺ DCs.

<p>(A) Six hr after LPS challenge, WT and GR^CD11c-cre splenocytes were analyzed for intracellular IL-12 levels. The right panels are histograms of CD8 staining of CD11c⁺ IL-12⁺ cells. (B) Intracellular IL-12 levels in CD8⁻ and CD8⁺ DCs between WT (shaded) and GR^CD11c-cre (solid line). One representative pair of a group of three is shown. (C) GC suppression of IL-12 production by CD8⁺ DCs ex vivo. Splenocytes were prepared from mice 3 hr after LPS injection and cultured with Brefeldin A in the presence or absence of corticosterone for 4 hr. Intracellular IL-12 levels on gated CD8⁺ DCs from one experiment are shown. The data are representative of three independent pairs of mice.</p

LPS-induced loss of CD8⁺ DCs is GR-dependent.

<p>(A–C) Splenic DCs from WT and GR^CD11c-cre B10.A mice were analyzed by flow cytometry at indicated times after injection with LPS (3 μg/g body weight). Only B220⁻TCRβ⁻ cells are shown. Results shown are from one representative experiment of three independent ones, each with three to five mice per group (D) Percentage of CD8⁺ DCs in WT and GR^CD11c-cre mice at time 0 (steady state). Results shown are from three pooled independent experiments, with 3–4 mice per group. (E) Total number of CD8⁺ and CD8⁻ DCs at time 0, 6 hr, and 24 hr post LPS injection. Results shown are from one representative experiment of three independent ones, each with three to five mice per group. (F) Sensitivity of DC subsets to GC-induced death in vitro. Total splenocytes from WT and GR^CD11c-cre mice were incubated with 100 nM corticosterone in vitro for 6 hr, and the percentage of dead cells in indicated populations are shown. The data shown are representative of four pairs of mice in two independent experiments. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

GR^CD11c-cre mice are more susceptible to LPS-induced septic shock than WT.

<p>(A) and (B) Immunoblot for GR in sorted DCs and other immune subsets from WT and GR^CD11c-cre mice. β-Actin was blotted as loading control. Some lanes were reordered for clarity, which is indicated by vertical white lines. (C) Plasma corticosterone concentrations after LPS challenge. Age- and sex-matched WT and GR^CD11c-cre mice were injected with LPS (3 μg/g body weight), and blood was drawn at indicated time points. Data shown are from four to eight animals per strain per time point pooled from multiple independent experiments. (D) GR^CD11c-cre mice (<i>n</i> = 4–6 per strain for each dose of LPS) were injected with the indicated amount of LPS, surface body temperature was recorded at the indicated times. * Four out of five GR^CD11c-cre mice died by 24 hr after LPS challenge. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p

GR does not change its oligomerization state in the presence of NF-κB.

<p>3617 cells transiently expressing the indicated combination of mCherry, mCherryGRwt, GFP, and/or GFP-P65 were treated with 300 nM Cort and/or 10 ng/ml TNF-α (TNF). (A) Fold-increase of the nuclear brightness (ε) relative to the control (total <i>n</i> = 215) for the green or red channels (color coded). Parenthesis means “in the presence of.” Bars with different superscript letters are significantly different from each other (<i>p</i><0.05). (B) subcelluar distribution of eGFP of mCherry in one representative cell for each condition. Scale bar = 10 µm. (C–D) Cross correlation analysis of the fluorescence fluctuations. The cross correlation brightness (Bcc) plot for each pixel (blue dots) of a representative nucleus as defined in Digman and colleagues <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001813#pbio.1001813-Digman2" target="_blank">[35]</a> as well as a histogram of the Bcc are shown.</p

The N&B method and its application to GR dimerization.

<p>(A) The molecular brightness (ε) can be measured by analyzing the frame-to-frame intensity fluctuations in the confocal volume (∼fl). Fluctuations are analyzed by measuring the ratio between the variance of the intensity signal σ² and the mean intensity value <i>. When fluorescent molecules are immobile, this ratio describes instrument noise which follows a Poisson distribution (σ² = <i>). If the signal fluctuates due to mobile molecules, the ratio σ²/<i> is directly proportional to the ε of the diffusing species. The scheme illustrates the standard deviation for an equal number of fluorophores organized as immobile monomers (σ0), mobile monomers (σ1), dimers (σ2), or trimers (σ3). Figure adapted from Hellriegel and colleagues <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001813#pbio.1001813-Hellriegel1" target="_blank">[55]</a>. The sampling time (i.e., pixel dwell time) has to be short enough that the intensity fluctuations are not averaged out (∼µs) while the re-sampling time (i.e., frame time) must be longer (∼ms-s) to measure independent fluctuations due to different populations of molecules at the same pixel <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001813#pbio.1001813-Digman1" target="_blank">[23]</a>. (B) To obtain ε of a fluorescent protein in living cells, a stack of images is acquired. Pixels that correspond to a specific region of interest (ROI) can be selected (e.g., the nuclear compartment) and ε is calculated for each pixel. Finally, the average ε for that region is obtained by fitting to a Gaussian distribution. (C–D) BHK cells (transfected with pEGFP-GR or pEGFP) were incubated with vehicle (control), 100 nM dexamethasone (Dex), 100 nM Cort or 10 µM CpdA. Where indicated, ligand was removed by washing and changing to ligand-free media (washout) as previously described <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001813#pbio.1001813-Stavreva1" target="_blank">[27]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001813#pbio.1001813-Liu1" target="_blank">[56]</a>. For each cell (<i>n</i> = 273) ε was calculated as shown in (B). (C) Fold-increase of the nuclear brightness (ε) relative to the control (monomeric GR). Means ± SEM are shown. Bars with different superscript letters are significantly different from each other (<i>p</i><0.05). (D) Subcelluar distribution in one representative cell for each treatment. Scale bar = 8 µm.</i></i></i></p

Transcriptional activity of the GR mutants.

<p>(A–B) Cos-7 cells were co-transfected with pEGFP-GR vectors and the MMTV-Luciferase reporter vector (A) or pkB-Luciferase reporter and pRelA expression vectors (B). Cells were incubated in the presence or absence of 100 nM Dex and luciferase activity was measured. (A) Values (induction efficiency) were expressed as percentage activity relative to Dex-treated wild-type GR. (B) Values (GR's inhibition efficiency on NF-κB activity) were expressed as percent induction relative to the control. (C) Nascent mRNA levels on MEFs cell lines stably expressing the GFPGR mutants. Means ± SEM from three independent experiments are shown. Bars with different superscript letters are significantly different from each other (<i>p</i><0.05).</p

DNA binding and chromatin recruitment of the GR mutants.

<p>(A) GR loading at the MMTV promoter array (white arrow) in the 3617 cell line (single cell analysis). Scale bar, 4 µm. (B–E) ChIPs using a GFP antibody in the 3134 cell line previously transfected with pEGFP-GR mutants. qPCR data as ChIP pulldown/input normalized to vehicle-treated cells (<i>n</i> = 4) for the MMTV promoter array (B) or endogenous GR binding sites, either <i>de novo</i> (C), pre-programmed (D), or nGRE (E). Means ± SEM from four independent experiments are shown. Bars with different superscript letters are significantly different from each other (<i>p</i><0.05). If at least one superscript letter is shared between treatments, then no significant differences were found.</p