Regulatory dendritic cells act as regulators of acute lethal systemic inflammatory response

Dendritic cells (DCs), the most potent antigen (Ag)-presenting cells (APCs), are defined by their dendritic morphology and unique phenotype and consist of heterogeneous subsets that belong to a myeloid or lymphoid lineage and have differing maturity in both lymphoid and peripheral tissues.¹  Immature DCs (iDCs) capture and process Ag in inflammatory tissues, and they develop into mature DCs (mDCs) with up-regulation of major histocompatibility complex (MHC) and costimulatory molecules in inflammatory microenvironments.¹  Subsequently, mDCs home into secondary lymphoid tissues, where they present the processed Ags to naive T cells to effectively generate effector T cells.¹  In addition, DCs also have the capacity to induce type-1 helper T (T_H1) cell and T_H2 cell responses, depending on their lineage and activation signals.¹  Thereby, DCs play a crucial role in linking between innate and adaptive immunity.¹ 

Recent studies suggest that iDCs play a crucial role in the induction of tolerance through the generation of anergic T cells and regulatory T (T_R) cells as well as peripheral deletion of T cells under steady state conditions in vivo.²  Treatment of iDCs with certain immunosuppressive molecules, such as interleukin 10 (IL-10), leads to the generation of tolerogenic DCs that show down-regulation of MHC and costimulatory molecules, and not only show defective T-cell activation but also possess tolerogenic properties.^3,4  In addition, several types of murine tolerogenic DCs naturally exist and/or are generated in secondary lymphoid areas under certain conditions.^4-6 

Host innate immune responses to bacterial infections are mediated primarily by conventional DCs and macrophages.^1,7  These cells sense the presence of invading pathogens via various pattern recognition receptors (PRRs) and stimulation of the PRR signaling pathway that initiates the secretion of proinflammatory mediators, which promote host inflammatory responses, resulting in the elimination of microorganisms.⁷  Despite a good understanding of the process that initiates and promotes host inflammation, little is known about the host immune cells responsible for the inhibition of the inflammatory response.

Sepsis is one representative type of systemic inflammatory response syndrome (SIRS) and is a major cause of morbidity and mortality in neonatal and medical intensive care units.⁸  Sepsis results from excessive stimulation of the host immune system by lipopolysaccharide (LPS)/endotoxin and other pathogen components to produce various proinflammatory cytokines and chemical mediators, and their overproduction can cause a systemic inflammation that in the worst case produces the pathological outcome of lethal sepsis.⁸  Despite continuing progress in the development of antibiotics and other supportive care therapies, there is a lack of effective means of prevention of or therapy for sepsis.^8,9  Indeed, therapies directed at neutralizing LPS or proinflammatory cytokines can prevent the development of septic shock in animal models, but clinical trials of these therapies have in general failed to improve the outcome of patients with sepsis.^8,9  Therefore, there is great enthusiasm about the development of novel strategies for the treatment of sepsis.

To address the mechanism responsible for the control of the host innate immune response, we examined the potential role of DCs in host inflammation using the models of murine experimental endotoxemia and bacterial peritonitis.

The preparation of DCs was described previously.¹⁰  Briefly, iDCs were prepared by culturing bone marrow (BM) cells (2 × 10⁵/mL) obtained from C57BL/6 mice, BALB/c mice (Charles River Laboratories, Raleigh, NC), or Il10KO mice (Jackson Laboratory, Bar Harbor, ME) with murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng/mL; Wako Pure Chemical Industries, Osaka, Japan) for 8 days. Regulatory DC precursors (pDC_regs) were generated from BM cells (2 × 10⁵/mL) cultured with murine GM-CSF (20 ng/mL), murine IL-10 (20 ng/mL, Wako Pure Chemical Industries), and human transforming growth factor (TGF)-β1 (20 ng/mL, Wako Pure Chemical Industries) for 8 days. Subsequently, iDCs or pDC_regs were washed 3 times with cold phosphate buffered saline (PBS) after the generation to prevent carryover of cytokines and were cultured with LPS (1 μg/mL; 055:B5, Sigma, St Louis, MO) for 2 days to generate mDCs or DC_regs, respectively.

Preparation of in vitro-generated and splenic CD11c^lowCD45RB^high DCs was performed as described in a previous report⁴  with some modifications. For preparation of in vitro-generated CD11c^lowCD45RB^high DCs, BM cells (2 × 10⁵/mL) were cultured with murine GM-CSF (20 ng/mL) and murine IL-10 (20 ng/mL) for 8 days. Subsequently, cells were positively selected with phycoerythrin (PE)-conjugated anti-mouse CD45RB monoclonal antibody (mAb) (BD Biosciences, San Diego, CA) and R-PE Magnetic Particles-DM (BD Biosciences). Based on the phenotype of in vitro-generated CD11c^lowCD45RB^high DCs, splenic CD11c^lowCD45RB^high DCs were obtained as follows. Splenic DCs were negatively selected from splenocytes using mouse Dendritic Cells Enrichment Set-DM (BD Biosciences). Splenic CD11c^lowCD45RB^high DCs were then negatively selected from purified splenic DCs with biotinylated anti-mouse CD11c mAb (BD Biosciences) plus IMag streptavidin Particles Plus-DM (BD Biosciences). The purity of CD11c^lowCD45RB^high cells was more than 90%, as indicated by FACS analysis, and fewer than 2 × 10⁵ cells/mouse of these subsets were collected.

For isolation of peritoneal macrophages, mice were given intraperitoneal injections of 1 mL 1% thioglycollate (DIFCO Becton Dickinson, Sparks, MD). Peritoneal exudate cells (PECs) were isolated from the peritoneal cavity 4 days after injection. Macrophages were positively selected from PEC with PE-conjugated anti-mouse CD11b mAb (BD Biosciences) and R-PE Magnetic Particles-DM. The purity of F4/80⁺CD11b⁺ cells was more than 90%, as indicated by FACS analysis (data not shown).

CD4⁺ T cells were negatively selected from spleen mononuclear cells with mouse CD4 T lymphocyte Enrichment Set-DM (BD Biosciences).

For stimulation of cells, DCs (5 × 10⁵) were stimulated or not stimulated with LPS (1 μg/mL) in the presence or absence of 8-Br-cyclic AMP (cAMP) (200 μM; Sigma) or Rp-8-Br-cAMPS (1 mM; Calbiochem, San Diego, CA) for 6 or 24 hours in 24-well plates (Becton Dickinson, Franklin Lakes, NJ). In another experiment, macrophages (5 × 10⁵) were stimulated or not stimulated with LPS (1 μg/mL) in the presence or absence of DCs (1.25 × 10⁵ to 5 × 10⁵), control Ig (10 μg/mL; Chemicon International, Temecula, CA), or anti-IL-10 Ab (10 μg/mL; R&D Systems, Minneapolis, MN) for 24 hours. The culture supernatants were collected and stored at -80°C until assayed for cytokines.

Cells were stained with fluorescein-conjugated mAbs to mouse CD14, CD40, CD45RB, CD80, CD86, I-A/I-E, or TLR4-MD2 or with isotype-matched control mAb (all from BD Biosciences). Analysis of fluorescence staining was performed with a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) and CELLQuest Software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Total RNA from DCs (10⁶) was extracted with TRIzol (Life Technologies, Gaithersburg, MD), and cDNA was synthesized with random hexamers as primer using the Superscript kit (Life Technologies). Expression levels of IκBNS¹¹  and Bcl-3¹¹  were measured by real-time polymerase chain reaction (PCR) (7300 Real Time PCR System, Applied Biosystems, Foster City, CA) using TaqMan universal PCR Master Mix (Applied Biosystems) and TaqMan probes mix for IκBNS and Bcl-3 (Applied Biosystems) after normalization for the expression of β-actin.

Cytokines in culture supernatants or sera were measured with ELISA kits (BioSource International, Camarillo, CA).

The concentration of intracellular cAMP was determined by enzyme immunoassay (Amersham Biosciences, Piscataway, NJ).

We used C57BL/6 mice (5 animals in each group) for murine experimental endotoxemia and bacterial peritonitis.^12-14  For LPS-induced endotoxemia, mice were given intraperitoneal injections of a mixture of LPS (1μg/mouse) and D-galactosamine (D-GalN, 10 mg/mouse; Sigma) 2 hours after the intraperitoneal injection of DCs (10⁵ to 10⁶/mouse). Alternatively, mice were given intraperitoneal injections of DCs (10⁶/mouse) 2 hours after intraperitoneal injection of LPS (1 mg/mouse). For bacterial peritonitis, mice were given intraperitoneal injections of DCs (10⁶/mouse) 2 hours after intraperitoneal injection of heat-killed (95°C for 30 minutes) Escherichia coli (DH5α, Invitrogen, Carlsbad, CA; 5 × 10⁸/mouse). For cecal ligation and puncture (CLP), mice were anesthetized with pentobarbital (1 mg/mouse, intraperitoneally), a small abdominal midline incision was made, and the cecum was exposed. The cecum was mobilized and ligated, and punctured through both surfaces once with an 18-gauge needle, and the abdomen was closed. Mice were given intraperitoneal or intravenous injections of DCs (10⁶/mouse) with or without control Ig (1 mg/mouse) or anti-IL-10 Ab (1 mg/mouse) 6 hours after CLP. In another experiment, CLP-treated mice were given intraperitoneal injections of DCs (10⁶/mouse) obtained from Il10KO mice. Survival was monitored at the indicated times for 48 hours (D-GalN-sensitized endotoxemia and bacterial peritonitis) or once daily for 6 days (endotoxemia and CLP). In some experiments, D-GalN-sensitized mice were killed at the indicated time after LPS challenge to obtain blood. The blood was processed to obtain serum and stored at -80°C until it was assayed for cytokines.

Cells (5 × 10⁶) were stimulated or not stimulated with LPS (1 μg/mL) for 10 minutes or 60 minutes and lysed with cell lysis buffer (Cell Signaling Technology, Beverly, MA) or NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL). The lysates or nuclear extracts were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Daiichi Pure Chemicals, Tokyo, Japan). After transfer to polyvinylidenefluoride (PVDF) membranes (Millipore, Bedford, MA) and subsequent blocking, the immunoblotting of membranes was performed with a PhosphoPlus p44/42 mitogen-activated protein kinases (MAPKs) (Thr202/Tyr204) antibody kit, a PhosphoPlus p38 MAPK (Thr180/Tyr182) antibody kit, a PhosphoPlus IκB-α (Ser32) antibody kit (all from Cell Signaling Technology) according to the manufacturer's instructions or anti-NF-κB p65 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) plus horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling Technology). Blot was visualized with a LumiGLO system (Cell Signaling Technology).

Thymocytes were prepared from tissue sections from the thymuses of mice 24 hours after CLP. The total number of thymocytes was measured by the trypan blue dye exclusion test, and apoptosis of thymocytes was assessed by flow cytometry using an annexin V-FITC Apoptosis Detection kit (R&D Systems).

CD4⁺ T cells (10⁵) obtained from normal or CLP-treated mice (24 hours after CLP) were cultured in 96-well plates (Becton Dickinson) with various numbers (1.25 × 10³ to 10⁴) of irradiated (15 Gy from a ¹³⁷Cs source, Gammacell 40 Exactor; MDS Nordion, Kanata, ON, Canada) allogeneic mDCs. [³H]thymidine (Amersham Biosciences, Piscataway, NJ) incorporation was measured on day 3 for the last 18 hours. In another experiment, the culture supernatants were collected and stored at -80°C until being assayed for cytokines.

The statistical significance of differences was determined by the Student paired t test or the log-rank test. P values below .01 were considered significant.

We previously established the methods to obtain murine DC_regs, which expressed moderate levels of MHC molecules but low levels of costimulatory molecules as compared with their normal counterparts (Figure 1A) and showed that they are more potent tolerogenic DCs than the previously known tolerogenic DCs with regard to the regulation of T-cell-mediated immune responses through the induction of anergic T cells and T_R cells in vivo and in vitro.¹⁰ 

CD14 and/or TLR4-MD2 complex act as receptors for LPS on macrophages and/or DCs.^7,15  We therefore examined the expression of TLR4-MD2 complex on pDC_regs and DC_regs (Figure 1B). Flow cytometric analysis showed that iDCs and pDC_regs expressed similar levels of TLR4-MD2 complex, but they did not express CD14. In addition, the expression levels of TLR4-MD2 complex on mDCs and DC_regs were reduced as compared with those of iDCs and pDC_regs.

We next examined the LPS-induced cytokine production of normal DCs and DC_regs (Figure 1C). Stimulation of iDCs with LPS caused vigorous production of TNF-α, IL-1β, IL-6, and IL-12p40, whereas this treatment induced significantly lower production of these proinflammatory cytokines in pDC_regs. mDCs constitutively produced large amounts of IL-6 and IL-12p40, but not TNF-α or IL-1β. In addition, mDCs showed slightly lower LPS-induced production of IL-6 and IL-12p40 than iDCs, whereas their production of TNF-α and IL-1β was significantly reduced compared with that of iDCs. In contrast, DC_regs exhibited defective production of LPS-induced proinflammatory cytokines. However, pDC_regs and DC_regs showed higher production of IL-10 than their counterparts.

Stimulation of DCs with LPS caused activation of MAPKs as well as NF-κB, and these events resulted in the functional activation of the DCs.⁷  To clarify the differences in the activation of the downstream signaling events of TLR4-MD2 complex on normal DCs and DC_regs, we examined the phosphorylation of Erk1/2 and p38 (Figure 1D). Stimulation with LPS induced higher levels of phosphorylated Erk1/2 (pErk1/2) and p38 (pp38) in iDCs than mDCs. In addition, the expression levels of both pErk1/2 and pp38 were significantly lower in pDC_regs and DC_regs than in iDCs and mDCs.

To detect the activation of NF-κB, we examined the expression of phosphorylated IκB-α (pIκB-α) and nuclear translocation of p65 (Figure 1D). iDCs showed higher levels of pIκB-α and nuclear translocation of p65 than mDCs when they were stimulated with LPS. In addition, pDC_regs and DC_regs showed significantly lower expression level of pIκB-α and nuclear translocation of p65 than their counterparts following stimulation with LPS.

Previous studies have shown that the IL-10-inducible expressions of a member of the IκB protein family, IκBNS and Bcl-3, interfered with the binding of NF-κB p50/p65 to the specific promoters of the targeted proinflammatory cytokines mediated through the interaction with NF-κB p50, and these IκB proteins are required for the IL-10-mediated suppression of proinflammatory cytokines in macrophages.¹¹  We therefore examined the transcriptional expressions of IκBNS and Bcl-3 in normal DCs and DC_regs (Figure 1E). Real-time PCR analysis showed that the expression level of IκBNS and Bcl-3 was higher in pDC_regs and DC_regs than iDCs and mDCs.

Various cAMP-elevating agents have been demonstrated to not only suppress TNF-α synthesis but also enhance IL-10 production in certain cell types.^16-18  To clarify the preferential production of IL-10 by pDC_regs and DC_regs, we measured the endogenous intracellular concentration of cAMP (Figure 1F). mDCs showed slightly higher amount of cAMP than iDCs, and stimulation with LPS had minimal effect. In contrast, both pDC_regs and DC_regs had increased amounts of cAMP compared witih iDCs and mDCs, and the stimulation with LPS caused further enhancement of the amount of cAMP.

To determine the role of protein kinase A (PKA) on the enhancement of IL-10 synthesis, since PKA is one of the molecular targets of cAMP, we examined the effect of the specific activator (8-Br-cAMP) or inhibitor (Rp-8-Br-cAMPS) of PKA on LPS-induced production of IL-10 in normal DCs and DC_regs (Figure 1G). Treatment of these DCs with 8-Br-cAMP increased LPS-induced production of IL-10, whereas Rp-8-Br-cAMPS decreased their production.

We tested the effect of DC_regs on LPS-induced proinflammatory production in mice with experimental endotoxemia (Figure 2). Intraperitoneal administration of a low dose of LPS (1 μg/mouse) induced transient high production of TNF-α, IL-1β, IL-6, and IL-12p40 in the serum in D-GalN-sensitized mice. A single intraperitoneal injection of iDCs 2 hours before injection of LPS had little or no effect on the serum production of these proinflammatory cytokines, whereas this treatment slightly enhanced the production of serum IL-10. In addition, a single intraperitoneal injection of mDCs 2 hours before injection of LPS slightly reduced the serum production of TNF-α, IL-1β, and IL-6, whereas this treatment enhanced the production of serum IL-12p40. In contrast, a single intraperitoneal injection of DC_regs showed more potent suppression of LPS-induced serum proinflammatory cytokine production than injection of pDC_regs, whereas a single intraperitoneal injection of pDC_regs caused higher production of serum IL-10 than injection of DC_regs.

To clarify the regulatory role of DC_regs in the inflammatory response in vivo, we examined the effect of DC_regs on septic shock induced by experimental endotoxemia and bacterial peritonitis. A single intraperitoneal injection of mDCs 2 hours before injection of a low dose of LPS slightly reduced the lethality in D-GalN-sensitized mice (Figure 3A). In contrast, a single intraperitoneal injection of DC_regs 2 hours before the injection of LPS potently prevented D-GalN-sensitized mice from LPS-induced lethality, and the preventive effect occurred in a dose-dependent fashion (Figure 3B). We also observed that DC_regs derived from other strains of mice exerted a potent preventive effect against LPS-induced lethality in D-GalN-sensitized mice, whereas mDCs derived from other strains of mice showed a minimal effect (data not shown).

To test the therapeutic effect of DC_regs against endotoxemia, mice were administered a high dose of LPS (1 mg/mouse) intraperitoneally and then treated with DC_regs, and the survival period was monitored. Treatment with DC_regs 2 hours after LPS challenge protected mice from the lethality, whereas treatment with mDCs had no effect (Figure 3C).

We also examined the therapeutic effect of DC_regs against lethality caused by bacterial peritonitis (Figure 3D). DC_regs showed a more potent protective effect against the lethality induced by bacterial peritonitis than mDCs when mice were given intraperitoneal injections of mDCs or DC_regs 2 hours after intraperitoneal injection of heat-killed E coli.

We further evaluated the therapeutic efficacy of DC_regs against polymicrobial sepsis induced by CLP, a model that more closely mimics the clinical scenario.^11,12  A single intraperitoneal or intravenous injection of DC_regs even 6 hours after CLP showed a significant protective effect against CLP-induced lethality, whereas treatment with mDCs had no effect (Figure 4A,B).

These findings indicate that DC_regs preferentially produced IL-10 in response to LPS (Figure 1C). We therefore examined the role of IL-10 in the protective effect of DC_regs against the lethality induced by CLP. Simultaneous injections of DC_regs and anti-IL-10 Ab, but not control Ig, inhibited the protective effect of DC_regs against CLP-induced lethality (Figure 4C). Moreover, treatment with DC_regs obtained from Il10KO mice¹⁹  showed a weaker protective effect against CLP-induced lethality than DC_regs obtained from normal mice (Figure 4D).

DC_regs suppressed LPS-induced production of proinflammatory cytokines by macrophages mediated through IL-10.

To clarify the mechanism underlying the suppression of inflammatory responses by DC_regs, we examined the effect of DC_regs on the LPS-induced production of TNF-α and IL-12p40 by macrophages. Stimulation of macrophages with LPS caused higher production of TNF-α than stimulation of mDCs or DC_regs, whereas the LPS-induced production of IL-12p40 in mDCs was higher than that in macrophages and DC_regs (Figure 5A,B). The addition of mDCs to LPS-stimulated macrophages significantly reduced the production of TNF-α as compared with the production by LPS-stimulated macrophages alone (Figure 5A), whereas their addition additively enhanced the production of IL-12 p40 by LPS-stimulated macrophages (Figure 5B). In contrast, the addition of DC_regs to LPS-stimulated macrophages significantly inhibited the production of both TNF-α and IL-12 p40 (Figure 5A,B).

We also examined the role of IL-10 in the DC_regs-mediated suppression of LPS-induced production of TNF-α and IL-12p40 by macrophages. Treatment of macrophages and mDCs with anti-IL-10 Ab, but not control Ig, slightly enhanced the LPS-induced production of TNF-α and IL-12p40 (Figure 5A,B). On the other hand, treatment with anti-IL-10 Ab largely abrogated the suppressive effect of DC_regs on the production of TNF-α and IL-12p40 by LPS-stimulated macrophages (Figure 5A,B).

To examine whether IL-10 secreted by DC_regs plays a direct role in the suppressive effect of these cells, we tested the effect of DC_regs obtained from Il10KO mice on the production of TNF-α and IL-12p40 by LPS-stimulated macrophages. mDCs and mDCs obtained from Il10KO mice showed similar ability to produce TNF-α and IL-12p40, whereas DC_regs obtained from Il10KO mice produced higher levels of these proinflammatory cytokines in response to LPS than DC_regs (Figure 5C,D). Furthermore, DC_regs obtained from Il10KO mice showed a weaker suppressive effect against LPS-induced proinflammatory cytokine production by macrophages than DC_regs (Figure 5C,D).

Previous studies have shown that CD11c^lowCD45RB^high DCs in the spleen and lymph nodes, which were identical to in vitro-generated IL-10-induced tolerogenic DCs, secreted IL-10 after activation, and these subsets induced Ag-specific tolerance through the differentiation of T_R cells.^4,6  To address the role of naturally existing tolerogenic DC subsets in host inflammation, we examined the effect of splenic CD11c^lowCD45RB^high DCs as well as their in vitro-generated counterparts on LPS-induced inflammatory responses. Flow cytometric analysis showed that splenic and in vitro-generated CD11c^lowCD45RB^high DCs exhibited higher expression of CD45RB than DC_regs, whereas the expression level of CD11c was similar among these DC subsets (Figure 6A). In addition, these CD11c^lowCD45RB^high DCs showed lower expression of I-A/I-E than DC_regs, whereas their expression level of CD86 was higher than that of DC_regs (Figure 6A). On the other hand, both splenic and in vitro-generated CD11c^lowCD45RB^high DCs showed production of IL-10 similar to that of DC_regs, whereas these subsets exhibited higher production of TNF-α than DC_regs following stimulation with LPS (Figure 6B).

We also compared the inhibitory effect of DC_regs and CD11c^lowCD45RB^high DCs on LPS-induced cytokine production and lethality in D-GalN-sensitized mice. Both splenic and in vitro-generated CD11c^lowCD45RB^high DCs showed lower suppressive effect on LPS-induced serum production of TNF-α than DC_regs, whereas similar production of serum IL-10 was observed when both of these types of DCs were injected into mice 2 hours before the injection of LPS (Figure 6C). In addition, treatment with these CD11c^lowCD45RB^high DCs showed a preventive effect similar to that of treatment with pDC_regs on LPS-induced lethality, although this effect was weaker than that of treatment with DC_regs (Figure 6D).

Several studies have shown that increased lymphocyte apoptosis, especially in the thymus, contributes to sepsis-induced lethality, and that blockage of the apoptosis improves the survival of septic mice.¹²  We therefore examined sepsis-induced thymocyte apoptosis in CLP-treated mice (Figure 7A,B). As reported previously, the significant reduction of thymocytes and marked thymocyte apoptosis were observed in untreated mice 24 hours following CLP. In addition, treatment with DC_regs, but not mDCs, ameliorated the reduction of thymocytes and thymocyte apoptosis in CLP-treated mice.

It has been postulated that the systemic inflammatory response caused by sepsis leads to the global immune suppression clinically seen in sepsis syndrome⁸ ; however, the impact of sepsis on host T-cell responses remains unclear. We therefore examined the ability of CD4⁺ T cells to proliferate and produce cytokines in CLP-treated mice (Figure 7C,D). CD4⁺ T cells obtained from untreated mice 24 hours following CLP exhibited marked reduction of the proliferative response as well as of the production of IL-2 and IFN-γ when stimulated with allogeneic mDCs. DC_regs, but not mDCs, inhibited the sepsis-induced reduction of the ability of CD4⁺ T cells to proliferate and produce cytokines.

In the present work, we show that a group of tolerogenic DCs are responsible for the regulation of the host inflammatory response that is triggered by bacteria and their products in vivo and in vitro. Our data shed new light on the crucial role of tolerogenic DCs not only in regulating T-cell responses in acquired immunity but also in the damping of host local and systemic inflammatory responses to microbial pathogens in innate immunity.

In contrast to the insights provided by a number of reports about the responses of iDCs to LPS and their functional outcome, the responsiveness of mDCs to LPS remains to be characterized. The ability of mDCs to produce TNF-α and IL-1β in response to LPS was significantly down-regulated compared with that of iDCs. In addition, mDCs exhibited lower activation of MAPKs and NF-κB than iDCs following stimulation with LPS. It is well known that previous exposure of macrophages to LPS induces a hyporesponsive state to secondary stimulation with LPS in terms of proinflammatory cytokine production and that this “LPS tolerance” is associated with the down-regulation of LPS-induced signaling events involving MAPKs and NF-κB.²⁰  Therefore, our results suggest that “LPS tolerance” could be induced in mDCs when they were generated by stimulation of iDCs with LPS. On the other hand, mDCs exhibited constitutively high production of IL-6 and IL-12p40, and they also further responded to stimulation with LPS. These results imply that the maturation-associated changes of the cytokine production profile in conventional DCs might promote an Ag-specific immune response rather than acute inflammation during the bacterial infection.

Although some types of tolerogenic DCs reportedly produced a higher amount of IL-10 instead of the lower production of proinflammatory cytokines compared with normal DCs following certain stimulations,^3-6  the molecular regulation of these events remains unclear. We showed that both pDC_regs and DC_regs produced lower levels of proinflammatory cytokines after stimulation with LPS as compared with iDCs and mDCs, whereas they preferentially produced IL-10. In addition, DC_regs obtained from Il10KO mice showed higher production of LPS-induced proinflammatory cytokines than DC_regs obtained from normal mice, suggesting that autocrine production of IL-10 by DC_regs is partially involved in the defective production by these cells of proinflammatory cytokines. We also showed that pDC_regs and DC_regs exhibited similar expression of the TLR4-MD2 complex compared with their respective counterparts, whereas the activation of LPS-induced signaling events involving MAPKs and NF-κB were impaired in pDC_regs and DC_regs. In addition, pDC_regs and DC_regs showed potent expressions of IκBNS and Bcl-3 when compared with their counterparts, suggesting that these IκB proteins also participate in the suppression of the NF-κB-mediated production of proinflammatory cytokines. Furthermore, pDC_regs and DC_regs showed constitutively higher amounts of intracellular cAMP than their counterparts, and the blockage of cAMP-mediated activation of PKA dramatically abrogated their LPS-induced production of IL-10. These results suggest that cAMP-mediated activation of PKA is a necessary intermediate event for the preferential production of IL-10 in pDC_regs and DC_regs. Collectively, both IL-10 and TGF-β1 used in the generation of pDC_regs and DC_regs appear to specifically regulate the downstream signaling events of the TLR4-MD2 complex leading to the characteristic cytokine production profile.

Treatment of D-GalN-sensitized mice with DC_regs completely blocked the LPS-induced increase of the levels of proinflammatory cytokines. DC_regs also significantly suppressed the production of TNF-α and IL-12p40 by LPS-stimulated macrophages. In addition, analysis of the suppression with anti-IL-10 Ab and DC_regs obtained from Il10KO mice showed that IL-10, especially IL-10 produced by DC_regs, is crucially involved in the inhibitory effect of DC_regs on the production of these proinflammatory cytokines by LPS-stimulated macrophages. Indeed, treatment of D-GalN-sensitized mice with DC_regs obtained from Il10KO mice showed a reduced suppressive effect on the serum production of proinflammatory cytokines (data not shown). Therefore, our results suggest that DC_regs suppress the production of the proinflammatory cytokines by inflammatory macrophages through the production of IL-10.

We showed that the treatment of mice with DC_regs protected against the lethality induced by experimental endotoxemia and bacterial peritonitis, even when treatment was started after the onset of the disease, possibly through the inhibition of host proinflammatory cytokine production. Previous studies have shown that local production of adenovirus-transduced IL-10 in the thymus and the draining lymph nodes, but not the systemic injection of exogenous IL-10, improved the survival of experimental septic mice, and improvements in survival were associated with the suppression of T-cell apoptosis through the enhancement of Bcl-2 expression and the reduction of caspase-3 activity.^21,22  We showed that the blockage of IL-10 reduced the protective effect of DC_regs against lethality caused by polymicrobial sepsis in CLP-treated mice. Indeed, treatment of septic mice with DC_regs significantly reduced thymocyte apoptosis as well as the suppression of CD4⁺ T-cell function. We also observed carboxyfluorescein diacetate-succinimidyl ester (CFSE)-labeled DCs in the spleen following intravenous injection into mice.¹⁰  In addition, DC_regs exhibited higher response to CCL5 but lower response to CCL19 than mDCs, implying that they traffic like semimature DCs (Katsuaki Sato, unpublished data, January 2003). Therefore, our results suggest that the protective effect of DC_regs against sepsis may involve the inhibition of macrophage activation in the inflammatory tissues as well as the reduction of the thymocyte apoptosis and CD4⁺ T-cell functional suppression in secondary lymphoid areas mediated through IL-10. Collectively, our findings suggest that the use of DC_regs might have preventive and therapeutic potential for the treatment of sepsis as well as other microbe-mediated systemic and local inflammatory disorders.

Although conventional DCs as well as tissue macrophages actively participate in the elimination of microbial pathogens through the initiation and progression of innate and acquired immunity, the mechanisms responsible for the regulation of host inflammation under pathophysiological conditions have remained largely unknown. We showed that naturally existing tolerogenic DC subsets as well as their in vitro-generated counterparts, which resemble DC_regs in terms of preferential production of IL-10, suppressed the inflammatory response in vivo. It has been suggested that “conditioned DC subsets” that exist in mucosal tissues in organized lymphoid organs regulate host immune responses to commensal and pathogenic bacteria.²³  In addition, mice devoid of IL-10 expression and function in myeloid-lineage cells develop chronic enterocolitis.^19,24  These phenomena lead us to hypothesize that naturally existing tolerogenic DC subsets producing IL-10 exert regulatory activity toward host inflammatory cells, resulting in the suppression of local and systemic inflammatory responses. Further insight into the subset heterogeneity and functional diversity of DCs should contribute to a better understanding of the regulation of innate and acquired immunity under steady-state and pathological conditions.

We would like to thank A. Takeuchi and M. Yamamoto for excellent assistance.