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.     

Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway

BACKGROUND: Commensal gut bacteria are essential for the development and maintenance of the gut's immune system. Some bacteria strains, such as Lactobacillus and Bifidobacterium species, have been reported to provide protection from allergic and inflammatory bowel diseases. However, the interactions between these commensal bacteria and the immune system are largely unknown. OBJECTIVE: We studied the effects of a supernatant from the culture of B breve C50 (BbC50) on the maturation, activation, and survival of human dendritic cells (DCs). METHODS: DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50 supernatant (BbC50SN) or LPS for 2 days. RESULTS: BbC50SN induced DC maturation, with increase in CD83, CD86, and HLA-DR expression. We also showed, for the first time, that BbC50SN prolonged DC survival, with high IL-10 and low IL-12 production compared with that seen in LPS-DCs. Moreover, BbC50SN inhibited the effects of LPS on DCs, both in terms of IL-12 production and in terms of survival. The prolonged DC survival was independent of IL-10 production and nuclear factor kappaB pathway but was associated with an upregulation of Bcl-xL and Phospho-Bad. Finally, BbC50SN induced activation of Toll-like receptor 2 (TLR2)-transfected cells in contrast to TLR4-, TLR7-, and TLR9-transfected cells. CONCLUSION: The supernatant of B breve C50 can induce DC maturation and prolonged DC survival through TLR2, with high IL-10 production. These properties might correspond to a regulatory DC profile, which could limit the excessive TH1 response and control the excessive TH2 polarization observed in atopic newborns.     

TLR7/8 agonists impair monocyte-derived dendritic cell differentiation and maturation

Pathogen recognition by TLR activates the innate immune response and is typically followed by the development of an adaptive immune response initiated by antigen presentation. Dendritic cells (DC) are the most efficient APC and express diverse TLRs, including TLR7 and -8, which have been recently identified as targets for ssRNA recognition during viral infection. We have studied the effect of TLR7/8 agonists on DC differentiation and maturation from human monocytes. The synthetic agonist Resiquimod (R-848) or the physiological agonist ssRNA impaired monocyte differentiation to DC phenotypically and functionally. Induced expression of the nonclassical MHC molecules of the CD1 family in DC was inhibited at the protein and mRNA levels, and antigen acquisition was inhibited. Proinflammatory cytokine (including IL-6, IL-8, TNF-alpha, IL-1beta) and IL-10 production were induced during DC differentiation. Cross-talk between TLR4 and TLR7/8 was revealed as immature DC, which had been differentiated in the presence of R-848 were insensitive to LPS-mediated maturation and cytokine production but still induced allostimulation. These data lead us to suggest that ongoing viral activation of TLR7/8 could alter the adaptive immune response by modifying DC differentiation and by down-regulating DC responsiveness to a subsequent bacterial TLR4-mediated signal.     

Porin-incorporated liposome induces Toll-like receptors 2- and 6-dependent maturation and type 1 response of dendritic cell

Porin of Shigella dysenteriae was incorporated in liposome (PIL) and presented to mouse splenic dendritic cells (DC). PIL up-regulated Toll-like receptor (TLR) 2 and TLR6 on DC, showing that co-expression of the two TLRs is involved in recognition of porin. Detection of myeloid differentiating factor 88 (MyD88)-TLR2 complex confirmed interaction between the two for triggering the downstream signaling, which ultimately led to TLR2-dependent nuclear translocation of nuclear factor-kappa B. PIL-induced expression of MHC class II (I-Ab), CD40 and CD80 showed maturation of DC, whereas up-regulation of intercellular adhesion molecule-1 and CCR7 implicated the capacity of splenic DC to migrate. Induction of messenger ribonucleic acid for the chemokines, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted indicated a strong bias of PIL for type 1 polarization that was supported by the intracellular expression and release of tumor necrosis factor (TNF)-alpha and IL-12. Along with CD40 and CD80 expression, release of the cytokines of CD11c+ JAWS II cells was inhibited by TLR2 or simultaneous TLR2 and 6 knockdown showing that recognition of PIL by the two TLRs is essential for DC activation and type 1 polarization. The signaling pathway initiated upon recognition of PIL by the TLRs was MyD88 dependent as confirmed by inhibition of IL-6, TNF-alpha and IL-12 release of MyD88-knockdown JAWS II cells. The maturation and polarization of DC induced T(h)1 phenotype, as evident from proliferation, activation and IFN-gamma release of allogeneic CD4+ T cells in response to PIL-stimulated DC, thereby suggesting that the adjuvant activity of PIL can successfully bridge the innate and adaptive immunity.     

Type I and II interferons enhance dendritic cell maturation and migration capacity by regulating CD38 and CD74 that have synergistic effects with TLR agonists

The major limitation for the maturation of dendritic cells (DCs) using Toll-like receptor (TLR) agonists is their decreased ability to migrate into lymph nodes compared with conventional DCs. CD38 can be used as a multifunctional marker to modulate migration, survival and Th1 responses of DCs. CD74 has been shown to negatively regulate DC migration. The goal of this study was to investigate the combinations of TLR agonists and interferons (IFNs) that most effectively regulate CD38 and CD74 expression on DCs. Synergistic TLR agonist stimulation in combination with IFN-α and IFN-γ was the best method for regulating CD38 and CD74 expression and inducing the highest secretion of IL-12p70. An in vitro migration assay showed that DCs treated with this combination had significantly enhanced migratory ability, similar to that observed in cells expressing CD38, CD74 and CCR7. The results of this study suggest that an alternative maturation protocol in which two TLR ligands are combined with type I and II IFNs generates potent DCs that have both a high migratory capacity and high IL-12p70 production.     

Combined Toll-like receptor agonists synergistically increase production of inflammatory cytokines in human neonatal dendritic cells

Dendritic cells (DC) are thought to be responsible for the reduced ability of human newborns to induce protective T-helper 1 (Th1) immune responses. The key player in Th1 differentiation, interleukin-12 (IL-12), is primarily produced in response to Toll-like receptor (TLR) binding by adult DC but not by neonatal DC. The potential use of various TLR agonist combinations for initiating neonatal monocyte-derived DC to prime Th1 responses was investigated. Single TLR ligands induced maturation only in adult DC; neonatal DC matured with combined targeting of TLR3/TLR8 or TLR4/TLR8, based on the expression of maturation markers. Similarly, the synergistic effects of combined TLR ligands could also be shown with adult and neonatal cytokine production, but different expression patterns were noted. In particular, IL-12p70 was produced by neonatal DC exclusively after combined TLR stimulation. Surprisingly, it was found that supernatants of combined stimulated neonatal DC could induce interferon-gamma production in autologous na?ve T cells. Moreover, this interferon-gamma secretion was blocked by anti-IL-12p70 antibodies and increased after addition of recombined IL-12. In conclusion, these findings underline the differences between adult and neonatal DC and might suggest new strategies for promoting newborn Th1 immunity in response to pathogens and vaccine antigens.     

IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells

Dendritic cells (DCs) play an important role in innate and adaptive immune responses. In addition to their phagocytic activity, DCs present foreign antigens to na?ve T cells and regulate the development of adaptive immune responses. Upon contact with DCs, activated T cells produce large quantities of cytokines such as interferon-gamma (IFN-gamma) and interleukin (IL)-21, which have important immunoregulatory functions. Here, we have analyzed the effect of IL-21 and IFN-gamma on lipopolysaccharide (LPS)-induced maturation and cytokine production of human monocyte-derived DCs. IL-21 and IFN-gamma receptor genes were expressed in high levels in immature DCs. Pretreatment of immature DCs with IL-21 inhibited LPS-stimulated DC maturation and expression of CD86 and human leukocyte antigen class II (HLAII). IL-21 pretreatment also dramatically reduced LPS-stimulated production of tumor necrosis factor alpha, IL-12, CC chemokine ligand 5 (CCL5), and CXC chemokine ligand 10 (CXCL10) but not that of CXCL8. In contrast, IFN-gamma had a positive feedback effect on immature DCs, and it enhanced LPS-induced DC maturation and the production of cytokines. IL-21 weakly induced the expression Toll-like receptor 4 (TLR4) and translation initiation region (TIR) domain-containing adaptor protein (TIRAP) genes, whereas the expression of TIR domain-containing adaptor-inducing IFN-beta (TRIF), myeloid differentiation (MyD88) 88 factor, or TRIF-related adaptor molecule (TRAM) genes remained unchanged. However, IL-21 strongly stimulated the expression of suppressor of cytokine signaling (SOCS)-1 and SOCS-3 genes. SOCS are known to suppress DC functions and interfere with TLR4 signaling. Our results demonstrate that IL-21, a cytokine produced by activated T cells, can directly inhibit the activation and cytokine production of myeloid DCs, providing a negative feedback loop between DCs and T lymphocytes.     

Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.     

TLR4在HSP70_(EC-TCV)冲激的DC成熟过程中的作用

观察TLR4在Hca-F榄香烯复合瘤苗来源的HSP70(HSP70EC-TCV)冲激处理的小鼠骨髓来源的DC成熟过程中的作用。用rmGM-CSF和rmIL-4诱导小鼠骨髓来源的DC,以HSP70EC-TCV或加入抗TLR4抗体30 min后再用HSP70EC-TCV冲激处理DC,流式细胞仪检测DC的CD40和CD86表达,ELISA法检测DC上清中IL-12和T细胞上清中IL-2浓度,MTT法检测T细胞对Hca-F细胞的杀伤率。结果表明,抗TLR4抗体对DC的CD40和CD86表达无明显影响,但对HSP70EC-TCV诱导DC分泌IL-12和进而致敏的T细胞分泌IL-2及产生杀瘤功能有明显的抑制作用。TLR4信号通路参与HSP70EC-TCV诱导DC成熟过程。     

Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with unresponsiveness to imidazoquinolines

Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8 alpha(+) DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8 alpha(-) DC and PDC, but not CD8 alpha(+) DC as measured by survival ex vivo, up-regulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8 alpha(+) DC in recognition of certain mouse pathogens.     

Dendritic cells differentiated in the presence of a single-stranded viral RNA sequence conserve their ability to activate CD4 T lymphocytes but lose their capacity for Th1 polarization

Monocyte-derived dendritic cells (DCs) differentiate in the presence of Toll-like-receptor (TLR) ligands in the course of ongoing infections. A single-stranded RNA (ssRNA) sequence, corresponding to the sequence of the U5 region of human immunodeficiency virus type 1 RNA, was used to mimic viral activation of TLR7 in human DCs. We determined the effector potential of DCs differentiated in the presence of this ssRNA molecule (ssRNA-DCs). ssRNA-DCs phenotypically resembled mature DCs. In contrast, their capacity to allostimulate naive CD4(+) T cells resembled that of conventional immature DCs and could be increased by TLR4 stimulation. Th1 polarization of CD4(+) T cells and production of interleukin 12p70 (IL-12p70) by ssRNA-DCs were selectively abrogated in response to a late TLR4, but not in response to a CD40, maturation signal. Inhibition of p38 mitogen-activated protein kinase partially restored IL-12p70 secretion but did not restore Th1 polarization, whereas addition of exogenous IL-12 led to recovery of Th1 polarization. In contrast to lipopolysaccharide, ssRNA induced IL-12p70 production at the very earliest stages of DC differentiation, indicating a particular role for TLR7 in monocyte-derived DCs recently engaged in differentiation. These data demonstrate generation of phenotypically mature DCs with the ability to expand CD4(+) T lymphocytes lacking Th1/2-polarizing capacity.     

Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model

The in vitro Peripheral Tissue Equivalent (PTE) module is a three‐dimensional tissue‐engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll‐like receptor (TLR) agonists, including polyinosine‐polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1α/β, 6, 8 and 10 and tumour necrosis factor‐α. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100‐fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up‐regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE‐derived DC treated with Gardiquimod, a TLR‐7 agonist, significantly augmented anti‐tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33⁺) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen‐presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.     

Toll-like receptor agonists differentially regulate cysteinyl-leukotriene receptor 1 expression and function in human dendritic cells

BACKGROUND: Dendritic cells (DCs) acquire, during their maturation, the expression of the chemokine receptor CCR7 and the ability to migrate to lymph nodes in response to CC chemokine ligand 19 (CCL19). This migration is impaired in mice lacking the leukotriene (LT) C4 transporter and restored by addition of exogenous LTC4. OBJECTIVE: To define the role of LT in human DC function, we studied the expression and function of the cysteinyl-leukotriene (CysLT) receptors during DC differentiation from monocytes and subsequent maturation. METHODS: Receptor expression was measured by flow cytometry and real-time PCR. Responsiveness to LTD4 stimulation was assessed by calcium flux and chemotaxis. RESULTS: Maturation of DC with LPS, a classic Toll-like receptor 4 agonist, reduced CysLT receptor 1 (CysLT1) expression by 50%, whereas CysLT receptor 2 expression was increased. In contrast, the Toll-like receptor 3 agonist poly inosinic and cytidylic acid (polyI:C) had no effect on receptor expression. Downregulation of CysLT1 expression by LPS could not be mimicked by TNF-alpha alone or in combination with IL-1beta or IL-6. It was, however, prevented by inhibitors of COX and could be reproduced by a combination of TNF-alpha and prostaglandin E2. Immature DCs and DCs matured with polyI:C, but not with LPS, responded to LTD4 with a robust cytosolic calcium flux, which was prevented by the CysLT1 antagonist montelukast. LTD4 induced DC chemotaxis and enhanced DC migration in response to CCL19 in DCs matured with polyI:C, but only weakly in DCs matured with LPS. CONCLUSION: Our data suggest that human DCs may differentially respond to leukotriene, depending on their maturational stimuli. CLINICAL IMPLICATIONS: Our study demonstrates that some microbial agents can reduce the migration of dendritic cells in response to leukotrienes, with potential for differential involvement of these cells in allergic inflammation.     

DC-SIGN ligation greatly affects dendritic cell differentiation from monocytes compromising their normal function

DC-SIGN is a C-type lectin selectively expressed by certain types of DCs, including monocyte-derived DCs. Many reports have described the impact of DC-SIGN engagement with concomitant TLR signaling in tailoring of the DC maturation process, but so far, none has addressed the importance of DC-SIGN engagement during their differentiation from blood progenitors. We therefore examined the role of DC-SIGN engagement limited to the stage of IL-4-guided differentiation of DCs from human peripheral blood monocytes but not during maturation. We used two different anti-DC-SIGN antibodies with reported DC-SIGN-engaging activities. In cultures with DC-SIGN ligands, the resulting iDCs displayed abrogated expression of differentiation markers CD1a and DC-SIGN. Without further DC-SIGN activation, such DCs matured with low CD80/CD86 and high ILT3 expression, along with the appearance of macrophage marker CD14. Additionally, treated DCs indicated a tolerogenic potential by possessing a low, allostimulatory capacity and inducing na?ve, allogeneic CD4(+) T cells to produce low levels of IFN-γ. Upon activation, IL-10 production was greatly increased by such DCs; however, the use of IL-10-blocking antibodies could not completely reverse alternative DC activation. This suggests an alternative activation response that is a result of a different elementary state of DCs generated with concomitant ligation of DC-SIGN. During differentiation, IL-4-induced pSTAT6 was reduced by DC-SIGN ligands. Furthermore, during LPS-induced maturation, treated DCs displayed lowered activation levels of p38 MAPK, STAT1, as well as STAT6, compared with controls. Collectively, evidence is presented confirming a crucial role for DC-SIGN signaling in DC generation from monocytes.     

Type I interferon regulates respiratory virus infected dendritic cell maturation and cytokine production

Activation of dendritic cells (DCs) by viruses is critical for both innate and adaptive immune responses. In this report, we investigated the role of type I interferon (IFN) in the activation of DCs by respiratory syncytial virus (RSV). Using DCs from type I IFNR-/- mice, these studies indicate that maturation, including upregulation of co-stimulatory molecules and optimal cytokine production, by RSV infection was dependent on type I IFN receptor signaling. Subsequently, studies using DCs from wild type mice demonstrate that continued production of type I IFN during later stages of DC maturation could alter their activation profiles. IFN-alpha and IFN-beta were upregulated in DCs grown from bone marrow of wild type mice after infection with RSV. In order to determine their function in competent DCs, blocking antibodies were used to specifically inhibit IFN-alpha/beta . The data demonstrate that production of IFN-beta, but not IFN-alpha, in RSV-infected wild type DCs promotes chemokine production and toll-like receptor (TLR) expression, while limiting IL-12 production. The inhibition of IL-12p70 by IFN-beta correlated with suppressed IL-12p40 expression levels. Furthermore, the addition of recombinant IFN-beta potently inhibited IL-12p40 expression in mature DC subsets during RSV infection, while only the highest dose of IFN-alpha had any inhibitory effect. Together, our studies provide insight into the complex regulation of DC maturation and IL-12 production co-ordinated by type I interferons in RSV-infected dendritic cells, and demonstrate that type I IFN has specific roles depending upon the stage of DC maturation.     

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.     

The STATus of PD-L1 (B7-H1) on tolerogenic APCs

Expression by DCs of co-inhibitory molecules such as programmed death ligand-1 (PD-L1/B7-H1/CD274), a member of the B7 superfamily, is crucial for the downregulation of T-cell responses and the maintenance of immune homeostasis. Exposure of immature DCs to danger-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPs) generally results in their maturation and acquisition of immunostimulatory function. However, exposure of DCs to TLR ligands early during their differentiation can inhibit further differentiation and confer tolerogenic properties on these APCs. A report in this issue of The European Journal of Immunology reveals that early inhibition of human DC differentiation from blood monocytes by TLR agonists is associated with a tolerogenic phenotype and Treg generation. The tolerogenic function of these APCs is dependent on MAPK-induced IL-6 and IL-10 production, which drives STAT-3-mediated PD-L1 expression. These observations link IL-10 and IL-6 to PD-L1 expression, providing a new dimension to the anti-inflammatory properties of these cytokines. These findings also have implications for understanding the inherent function of DCs in non-lymphoid tissues such as the liver and lung, where they are exposed to PAMPs that are found constitutively in the local microenvironment.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.