Endotoxin can induce MyD88-deficient dendritic cells to support T(h)2 cell differentiation

Toll-like receptor (TLR) signaling activates dendritic cells (DC) to secrete proinflammatory cytokines and up-regulate co-stimulatory molecule expression, thereby linking innate and adaptive immunity. A TLR-associated adapter protein, MyD88, is essential for cytokine production induced by TLR. However, in response to a TLR4 ligand, lipopolysaccharide (LPS), MyD88-deficient (MyD88(-/-)) DC can up-regulate co-stimulatory molecule expression and enhance their T cell stimulatory activity, indicating that the MyD88-independent pathway through TLR4 can induce some features of DC maturation. In this study, we have further characterized function of LPS-stimulated, MyD88(-/-) DC. In response to LPS, wild-type DC could enhance their ability to induce IFN-gamma production in allogeneic mixed lymphocyte reaction (alloMLR). In contrast, in response to LPS, MyD88(-/-) DC augmented their ability to induce IL-4 instead of IFN-gamma in alloMLR. Impaired production of T(h)1-inducing cytokines in MyD88(-/-) DC cannot fully account for their increased T(h)2 cell-supporting ability, because absence of T(h)1-inducing cytokines in DC caused impairment of IFN-gamma, but did not lead to augmentation of IL-4 production in alloMLR. In vivo experiments with adjuvants also revealed T(h)2-skewed immune responses in MyD88(-/-) mice. These results demonstrate that the MyD88-independent pathway through TLR4 can confer on DC the ability to support T(h)2 immune responses.     

Toll-like receptor (TLR)2 and TLR3 synergy and cross-inhibition in murine myeloid dendritic cells

Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. Few studies have been devoted to address the impact of TLR2 (a fully MyD88-dependent receptor) and TLR3 (a fully TRIF-dependent receptor) co-activation on DC functions, especially in the mouse system. Using canonical agonists, we show that TLR2 acts in concert with TLR3 to induce the synthesis of inflammatory cytokines (TNF-alpha, IL-6), of some IL-12 family members (IL-12p40, IL-12p23, IL-27p28) and of the Notch ligand Delta-4 by mouse DCs. In contrast, TLR2 interferes with the TLR3-induced expression of type I interferon stimulated genes (MIG/CXCL9, IP-10/CXCL10, GARG39) and IL-12p35. We also report that TLR2 cooperates with TLR3 to enhance the DC-mediated production of IFN-gamma by Natural Killer cells and by conventional Ag-specific T lymphocytes. To conclude, our data support the existence of TLR2 and TLR3 synergy and cross-inhibition in DCs that could be important to strengthen immune responses during infection.     

The role of suppressor of cytokine signaling 1 as a negative regulator for aberrant expansion of CD8alpha+ dendritic cell subset

The suppressor of cytokine signaling (SOCS) 1 is a negative regulator in multiple cytokine-related aspects to maintain immunological homeostasis. Here, we studied a role of SOCS1 on dendritic cell (DC) maturation in the mice lacking either TCRalpha chain or CD28 in SOCS1-deficient background, and found that the SOCS1 could restore acute phase of inflammatory response in SOCS1-deficient mice. The CD11c+ CD8- DC population in freshly isolated splenic DCs from normal mice highly expressed SOCS1. However, in SOCS1-deficient environment, the proportion of CD8alpha+ DCs (CD8 DCs) noticeably increased without affecting the cell number of conventional and plasmacytoid DC populations. This population revealed the CD11cdull CD8alpha+ CD11b- CD45RA- B220- phenotype, which is a minor population in normal mice. Localization of the abnormal CD8 DCs in splenic microenvironments was mainly restricted to deep within red pulp. The CD8 DCs secrete a large amount of IFN-gamma, IL-12 and B lymphocyte stimulator/B cell activation factor of the tumor necrosis factor family in response to LPS and CpG stimulation. This is responsible for the development of DC-mediated systemic autoimmunity in the old age of SOCS1-deficient mice. Moreover, the CD8 DC subsets expressed more indoleamine 2,3-dioxygenase and IL-10, and hence inhibit the allogeneic proliferative T cell response and antigen-induced Th1 responses. Therefore, SOCS1 expression during DC maturation plays a role in surveillance in controlling the aberrant expansion of abnormal DC subset to maintain homeostasis of immune system.     

TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (TIR) domain-containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.     

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells

We report the effects of hemicellulase-treated Agaricus blazei (ABH) on the maturation of bone-marrow-derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up-regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T-cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin-12 (IL-12) p40, tumour necrosis factor-alpha, or IL-1beta. In addition, ABH suppressed LPS-induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS-induced cytokine secretion, while only slightly decreasing up-regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan-induced or CpG oligodeoxynucleotide-induced IL-12p40 production in DCs. The inhibition of LPS-induced responses was not associated with a cytotoxic effect of ABH nor with an anti-inflammatory effect of IL-10. However, ABH decreased NF-kappaB-induced reporter gene expression in LPS-stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS-stimulated but non-ABH-pretreated DCs. These observations suggest that ABH regulates DC-mediated responses.     

Lysophosphatidic acid modulates the activation of human monocyte-derived dendritic cells

Lysophosphatidic acid (LPA) is a biologically active lysophospholipid that can regulate immune activation. LPA can activate T cells and dendritic cells (DCs), but the effects of LPA on the ability of DCs to influence T cell polarization are not well understood. Human monocyte-derived DCs were differentiated in vitro in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colonystimulating factor (GM-CSF), and matured with LPA and lipopolysaccharide (LPS) alone or in combination. DC activation was monitored by analyzing cell-surface expression of co-stimulatory receptors and cytokine production. The ability of DCs to influence T cell activation was determined using two models of DC:T cell co-culture. Maturation with LPS induced dose-dependent DC activation characterized by enhanced expression of co-stimulatory molecules (e.g., CD86) and production of cytokines including IL-6 and IL-10. Co-incubation with LPA attenuated the LPS-induced production of IL-6, without significantly affecting IL-10 secretion or the ability of DC to promote T cell proliferation. DCs matured in the presence of both LPA and LPS enhanced the production of interferon-gamma (IFN-gamma) when co-cultured with allogeneic T cells, compared with DC matured by LPS alone. Similar results were found using a model of allogeneic na?ve T cell differentiation, where LPA- plus LPS-matured DC enhanced IFN-gamma as well as IL-4 secretion after restimulation. Lysophosphatidic acid fine-tunes the effects of LPS on human myeloid DC maturation, but does not exert a dominant effect on the ability of DC to influence Th cell polarization.     

Maturation of murine bone marrow-derived dendritic cells with poly(I:C) produces altered TLR-9 expression and response to CpG DNA

Although poly(I:C) and LPS induced differential dendritic cell (DC) cytokine profiles and toll-like receptor (TLR) expression, all were capable of causing phenotypic and functional DC maturation. Both LPS and poly(I:C) downregulated TLR-4/MD-2 expression on DCs. Although poly(I:C) highly upregulated their cell surface TLR-9 expression, LPS upregulated the intracellular TLR-9 expression. LPS-treated DCs could not produce IL-12p70 in response to subsequent both LPS- and CpG DNA-stimulation. On the other hand, poly(I:C)-treated DCs retained to produce IL-12p70 by subsequent CpG DNA-stimulation, while subsequent LPS-stimulation did not induce IL-12p70 production. Chloroquine, inhibitor of endosomal maturation, completely inhibited cytokine production of LPS-treated DCs as well as unstimulated control in response to subsequent CpG DNA-stimulation, while it failed to delete the IL-12p40 and IL-10 production in poly(I:C)-treated DCs. These data suggest that poly(I:C) may induce a novel DC phenotype that preserves the capacity of cytokine production to subsequent CpG DNA-stimulation.     

Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells

A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell (DC) cultures to examine possible T helper type 1 (Th1) responses induced by MDP and FK565/156 in combination with various synthetic Toll-like receptor (TLR) agonists, including synthetic lipid A (TLR4 agonist), the synthetic triacyl lipopeptide Pam3CSSNA (TLR2 agonist), poly(I:C) (TLR3 agonist), and CpG DNA (TLR9 agonist). Immature DCs derived from human monocytes expressed mRNAs for Nod1, Nod2, TLR2, TLR3, TLR4, and TLR9. The stimulation of DCs with MDP and FK565 in combination with lipid A, poly(I:C), and CpG DNA, but not with Pam3CSSNA, synergistically induced interleukin-12 (IL-12) p70 and gamma interferon (IFN-gamma), but not IL-18, in culture supernatants and induced IL-15 on the cell surface. In correlation with the cytokine induction, an upregulation of the mRNA expression of these cytokine genes was observed. Notably, IL-12 p35 mRNA expression increased >1,000-fold upon stimulation with lipid A plus either MDP or FK565 compared with stimulation with each stimulant alone. In contrast, for the expression of CD83 and costimulatory molecules such as CD40, CD80, and CD86, no synergistic effects were observed upon stimulation with Nod plus TLR agonists. The culture supernatants of DCs stimulated with lipid A plus either MDP or FK565 activated human T cells to produce high levels of IFN-gamma, and the activity was attributable to DC-derived IL-12. These findings suggest that Nod1 and Nod2 agonists in combination with TLR3, TLR4, and TLR9 agonists synergistically induce IL-12 and IFN-gamma production in DCs to induce Th1-lineage immune responses.     

Combinations of IFN-gamma and IL-4 induce distinct profiles of dendritic cell-associated immunoregulatory properties

Interferon gamma (IFN-gamma) and interleukin-4 (IL-4) are not only generated during cell-mediated immunity (CMI) and humoral immunity (HI), but are also generated by innate immune cells in response to pathogenic factors. How these cytokines differentially effect the development of dendritic cell (DC)-associated immunoregulatory properties from progenitor cells during innate immunity is unresolved. To address this we have utilized a homogeneous DC progenitor-like cell line, MTHC-D2, as a model to examine cytokine-induced maturation of DCs. By 6 h IFN-gamma induced genes that are important for antiviral activity and development of CMI, whereas IL-4 induced genes involved in cellular adhesion, uptake of extracellular antigen, suppression of cytotoxic T-cell responses, and that repair the extracellular matrix. By 48 h the cytokine stimulus had induced many properties characteristic of immature DCs; however, these were differentially effected by IFN-gamma and IL-4. IFN-gamma induced the greatest levels of costimulatory/ activation marker expression, and the highest levels of T-cell proliferation, whereas IL-4 induced the greatest levels of phagocytic activity. Stimulation of the cells with CD40 Ab enhanced the levels of costimulatory marker expression and T-cell stimulatory capacity of cells exposed to IFN-gamma, but had little effect on cells exposed to IL-4 in the absence of IFN-gamma.     

Engagement of human monocyte-derived dendritic cells into interleukin (IL)-12 producers by IL-1beta + interferon (IFN)-gamma

Dendritic cells (DCs) are potent antigen-presenting cells and can induce tumour- or pathogen-specific T cell responses. For adoptive immunotherapy purposes, immature DCs can be generated from adherent monocytes using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, and further maturation is usually achieved by incubation with tumour necrosis factor (TNF)-alpha. However, TNF-alpha-stimulated DCs produce low levels of IL-12. In this study, we compared the effects of TNF-alpha, interferon (IFN)-gamma, IL-1beta or IFN-gamma + IL-1beta on the phenotypic and functional maturation of DCs. Our results show that IFN-gamma, but not IL-1beta, augmented the surface expression of CD80, CD83 and CD86 molecules without inducing IL-12 production from DCs. However, IL-1beta, but not IFN-gamma, induced IL-12 p40 production by DCs without enhancing phenotypic maturation. When combined, IFN-gamma + IL-1beta treatment profoundly up-regulated the expression of CD80, CD83, CD86 and major histocompatibility complex (MHC) class II antigens. Furthermore, IFN-gamma + IL-1beta-treated DCs produced larger amounts of IL-12 and induced stronger T cell proliferation and IFN-gamma secretion in primary allogeneic mixed lymphocyte reaction (MLR) than did TNF-alpha-treated DCs. Our results show that IFN-gamma + IL-1beta induced human monocyte-derived DCs to differentiate into Th1-prone mature DCs.     

Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation.     

Dendritic Cells Transduced with SOCS1 Gene Exhibit Regulatory DC Properties and Prolong Allograft Survival

SOCS1 is a key regulator of cytokine signaling and is important for maintaining balance in the immune system. It is thought to participate in negative feedback loops in cytokine signaling and may be an important signal for the regulation of dendritic cell （DC） maturation. However, it remains unclear whether DCs transduced with SOCS1 exhibit characteristics of regulatory DCs and induce allogeneic T-cell hyporesponsiveness. In this study, we constructed adenovirai vector coding SOCS1 （Ad-SOCS1） that can efficiently increase SOCS1 gene expression in bone marrow-derived dendritic cells. DCs transduced with Ad-SOCS1 （DC-SOCS1） expressed low levels of costimulatory and MHC molecules, were resistant to maturation and activation stimulation, induced allogeneic T-cell hyporesponsiveness, and promoted the generation of regulatory-like T cells in vitro. DC-SOCS1 pretreatment significantly prolonged the survival of ailografts and led to a substantial increase in the generation of regulatory T cells. Our data suggest that SOCS1 inhibits DC maturation and induces regulatory DC generation, therefore possessing therapeutic potential to prevent rejection in organ transplantation.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells]

We investigated the role of galectin-9 (Gal-9) in maturation of dendritic cells (DC). Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a concentration-dependent manner, although Gal-9 had no effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2), but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4(+) T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were only slightly inhibited by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities, and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the p38 MAPK and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or phosphatidylinositol 3-kinase, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

Toll-like receptor signaling is impaired in dendritic cells from patients with X-linked agammaglobulinemia

Bruton's tyrosine kinase (BTK), which is defective in patients with X-linked agammaglobulinemia (XLA), is expressed not only in B cells but also in monocytes and dendritic cells (DCs). DCs play a crucial role in the innate immune response against infections by sensing pathogens through Toll-like receptors (TLRs). However, it is not known whether BTK deficiency in XLA might impair TLR-mediated signaling in DCs, which are susceptible to various infections. The phenotypic maturation and cytokine production mediated by TLRs were examined in monocyte-derived DC from XLA patients and normal controls. The TLR expression in DCs was analyzed by flow cytometry. TLR-mediated signaling in DCs was evaluated for the phenotypic maturation based on CD83 expression and production of cytokines, such as TNF-alpha, IL-6 and IL-12p70. TLR levels in DCs were similar between XLA and controls. TLR2, TLR4 and TLR7/8 ligands elicited less phenotypic maturation of DCs from XLA patients than normal controls based on CD83 expression. Stimulation with TLR2, TLR4 and TLR7/8 ligands, as well as TLR3 ligand, resulted in significantly lower production of TNF-alpha, but neither IL-6 nor IL-12p70, by DCs from XLA patients in comparison to normal controls. These findings suggest that BTK may thus be required for TLR signaling in DCs. The impaired TLR signaling in DCs may therefore be partly responsible for the occurrence of severe infections with bacteria and some viruses in XLA patients.     

Late dendritic cells are still able to evoke a potent alloreactive CTL response

On exposure to maturation stimuli, immature dendritic cells (DCs) undergo changes that turn them into potent amplifiers of innate immunity and into antigen-presenting cells (APCs) able to prime na?ve T cells. However, their progression through the maturation process is very rapid and finally ends in apoptosis. The aim of our study was to investigate the importance of the maturation stage of DCs, defined by morphology, expression of surface markers and IL-12 production, for their immunostimulatory capacity. DCs were matured with LPS, monocyte-conditioned medium (MCM) or TNF-alpha, sampled several times during a 3-day long maturation period and used as stimulators of allogeneic T cells over a wide range of DC/T cell ratios. T-cell response was assessed by cell proliferation, CTL generation and IFN-gamma production. Our results indicate that the in vitro T cell response is determined mainly by the level of expression of co-stimulatory molecules on DCs and the DC/T cell ratio in the culture. Thus, DCs matured for over 20h, with high expression of co-stimulatory molecules, can still induce a potent CTL response at DC/T cell ratios of 1:10 and 1:20, although their IL-12 production, as well as their ability to induce IFN-gamma production by T cells, are both decreased. In contrast, the CTL response at DC/T cell ratios of 1:2 and 1:5 can be profoundly decreased. Notably, the proportion of proliferating CD4+ T cells in these cultures is reduced. This could well be the reason for the absence of CTL response, since we showed that, even in the case of high expression of co-stimulatory molecules on DCs, generation of CTLs still depends on CD4+ T cells. Our study emphasizes the importance of strong expression of co-stimulatory molecules on DCs and of their ability to activate CD8+ and CD4+ T cells concomitantly in order to initiate a potent cell-mediated immune response. We therefore suggest that a combination of early DCs, which are strong producers of cytokines, and late DCs, which have high expression of co-stimulatory molecules, could prove beneficial in the attempt to initiate in vitro and in vivo cell-mediated immune responses for therapeutic purposes.     

Delivery of rapamycin by PLGA nanoparticles enhances its suppressive activity on dendritic cells

The purpose of this study was to evaluate the effect of rapamycin delivery by poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles on the maturation of dendritic cells (DCs). DCs were generated from mouse bone marrow and exposed to particulate and soluble rapamycin without any additional treatment, or with pre- or posttreatment with lipopolysaccharide (LPS). Annexin V-FITC/PI staining was performed on DC cultures to assess the viability of DCs during study. Surface phenotype of DCs was characterized for the expression of maturation markers, that is, MHC class II, CD86, and CD40 by flow cytometry. Cell culture supernatants were analyzed for the production of TGF-beta, IL-12, and IL-10 cytokines using sandwich ELISA method. DCs from Balb/C mice were cocultured with T cells from C57BL/6 mice and allogenic mixed lymphocyte reaction was assessed by [3H]-Thymidine incorporation. Unlike free rapamycin that has shown little if any effect on the expression of maturation markers in immature DCs, PLGA encapsulated rapamycin decreased the expression of all maturation markers under the study, that is, MHC class II, CD86, and CD40, significantly. LPS pre- or posttreated DCs demonstrated decreased expression of MHC class II, CD86, and CD40 in the presence of soluble or encapsulated rapamycin. The cytokine secretion profiles revealed high levels of TGF-beta and very low levels of IL-10 and IL-12 production. Rapamycin in soluble or encapsulated form significantly inhibited mixed lymphocyte reaction in DCs. The inhibitory effect of rapamycin on the maturation of DCs with respect to DC phenotype, cytokine production, and functional effects on the proliferation of T cells was significantly increased by PLGA delivery.     

Regulatory role of nitric oxide on monocyte-derived dendritic cell functions.

Nitric oxide (NO) has an established role in the defense against bacterial infections and exerts multiple modulatory activities on both inflammatory and immune responses. However, the relevance of NO on dendritic cell (DC) functions has been poorly investigated. In this study, we found that addition of the NO donor S-nitrosoglutathione (GSNO) to monocyte-derived DCs matured by lipopolysaccharide (LPS) or soluble CD40 ligand led to a decreased capacity to activate naive allogeneic T cells but a more prominent Th1 polarization, with increased interferon-gamma (IFN-gamma) secretion and reduced interleukin-5 (IL-5) release. The presence of GSNO during maturation of DCs caused a reduced expression of surface CD86, whereas CD80, CD83, and MHC molecule expression was not affected. Moreover, GSNO induced a dose-dependent decrease of IL-10 and enhancement of tumor necrosis factor-alpha (TNF-alpha) release from mature DCs. In parallel, a marked reduced production of IL-12 p40 subunit but no significant perturbation of the bioactive IL-12 p70 production was observed. Finally, GSNO significantly reduced the release of IP-10/CXCL10 and RANTES/CCL5 but not IL-8/CXCL8 by mature DCs. Although GSNO can strengthen the capacity of mature DCs to induce type 1 polarization of T lymphocytes, our data suggest that it elicits distinct anti-inflammatory functions, eventually reducing T lymphocyte proliferation and recruitment.     

CD40L stimulation of rat dendritic cells specifically favors the IL-12/IL-10 ratio resulting in a strong T cell stimulatory capacity

Dendritic cells (DC) play an important role in immune responses and have been studied extensively in human and mouse models. CD40 triggering of DC has a pivotal role in their maturation process, obtaining the unique capacity to induce strong CD4 and CD8 T cell activation. Although rat models are frequently used for the understanding of the underlying mechanism of human diseases, relatively little is known about rat DC. To investigate the effect of CD40 triggering on rat DC, we cloned the rat CD40L gene and generated murine fibroblasts with stable expression (L-rCD40L). DC stimulated by L-rCD40L cells exhibited a strong T cell stimulatory capacity, associated with higher amounts of IFN-gamma as compared to LPS-stimulated DC. Analysis of cytokine production showed that LPS induced both IL-12 and IL-10 production, whereas CD40L induced high amounts of IL-12, but little IL-10 production by rat DC. This implies that the difference found in T cell stimulatory capacity by the stimulated DC is due to the cytokine profile of the DC at the time of T cell activation.     

Commensal bacteria trigger a full dendritic cell maturation program that promotes the expansion of non-Tr1 suppressor T cells

Dendritic cells (DCs) orchestrate the immune response establishing immunity versus tolerance. These two opposite functions may be dictated by DC maturation status with maturity linked to immunogenicity. DCs directly interact with trillions of noninvasive intestinal bacteria in vivo, a process that contributes to gut homeostasis. We here evaluated the maturation program elicited in human DCs by direct exposure to commensal-related bacteria (CB) in the absence of inflammatory signals. We showed that eight gram(+) and gram(-) CB strains up-regulated costimulatory molecule expression in DCs and provoked a chemokine receptor switch similar to that activated by gram(+) pathogens. CB strains may be classified into three groups according to DC cytokine release: high IL-12 and low IL-10; low IL-12 and high IL-10; and low IL-12 and IL-10. All CB-treated DCs produced IL-1beta and IL-6 and almost no TGF-beta. Yet, CB instructed DCs to convert naive CD4+ T cells into hyporesponsive T cells that secreted low or no IFN-gamma, IL-10, and IL-17 and instead, displayed suppressor function. These data demonstrate that phenotypic DC maturation combined to an appropriate cytokine profile is insufficient to warrant Th1, IL-10-secreting T regulatory Type 1 (Tr1), or Th17 polarization. We propose that commensal flora and as such, probiotics manipulate DCs by a yet-unidentified pathway to enforce gut tolerance.