Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses.     

Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis

Dendritic cells (DCs) play a key role in the induction and regulation of antigen-specific immunity. Studies have shown that, similar to infection, cellular necrosis can stimulate DC maturation. However, the ability of necrotic cell death to modulate DC cytokine secretion has yet to be explored. We investigated the regulation of interleukin (IL)-12 secretion by human DCs in response to tumour cell necrosis in an in vitro culture model. Two human tumour cell lines (K562 and JAr) were induced to undergo necrosis using heat injury and repeated cycles of freezing and thawing. Both types of tumour cells tested in this study, when injured, induced secretion of monomeric IL-12p40 by monocyte-derived DCs. Furthermore, priming DCs with necrotic cells augmented IL-12p70 secretion significantly in conjunction with CD40 cross-linking. This was physiologically relevant because cell death-pulsed DCs were more potent than non-pulsed DCs at stimulating T cells to proliferate and secrete interferon (IFN)-gamma. The Toll-like receptor 4 (TLR4) played a role in mediating the DC response to heat-killed, but not freeze/thaw-killed necrotic cells. For both methods of injury, proteins contributed to the effect of necrosis on dendritic cells, whereas DNA was involved in the effect of freeze/thawed cells only. These findings indicate that necrotic tumour cell death is not sufficient to induce bioactive IL-12p70, the Th1 promoting cytokine, but acts to augment its secretion via the CD40/CD40L pathway. The results also highlight that the mode of cell death may determine the mechanism of dendritic cell stimulation.     

Activation of plasmacytoid dendritic cells by apoptotic particles – mechanism for the loss of immunological tolerance in Sjögren's syndrome

Sjögren's syndrome (SS) is a common autoimmune disease targeting salivary and lacrimal glands. It is strongly female‐dominant, characterized by low oestrogen levels combined with a local intracrine dihydrotestosterone defect. We hypothesized that these hormonal deficits lead to increased apoptosis of the epithelial cells and plasmacytoid dendritic cell (pDC)‐mediated proinflammatory host responses. Expression of Toll‐like receptors (TLRs)‐7 and ‐9 and cytokine profiles was studied in pDCs treated with apoptotic particles collected in consecutive centrifugation steps of media from apoptotic cells. Expression and localization of SS autoantigens in these particles was also analysed. Furthermore, the effects of sex steroids were studied in pDCs cultured with several concentrations of dihydrotestosterone and 17‐β‐oestradiol, and in saliva of patient treated with dehydroepiandrosterone. Apoptosis of the epithelial cells led to cleavage and translocation of SS‐autoantigens, α‐fodrin and SS‐A, into apoptotic particles. The apoptosis‐induced apoptotic particles also contained another SS‐autoantigen, hy1‐RNA. These particles were internalized by pDCs in a size‐dependent manner and affected TLR‐7 and ‐9 expression and the production of proinflammatory cytokines. The analysed androgens protected cells from apoptosis, influenced redistribution of autoantigens and diminished the apoptotic particle‐stimulated increase of the TLRs in pDCs. Our findings suggest that the formation of apoptotic particles may play a role in loss of immune tolerance, manifested by production of autoantibodies and the onset of autoinflammation in SS.     

Differential production of interleukin-10 and interleukin-12 in mononuclear cells from leprosy patients with a Toll-like receptor 2 mutation

Toll-like receptor 2 (TLR2) is a key mediator of the immune response to mycobacterial infections, and mutations in TLR2 have been shown to confer susceptibility to infection with mycobacteria. This study investigated the profiles of cytokines, such as interferon (IFN)-γ, interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α in response to Mycobacterium leprae in peripheral blood mononuclear cells (PBMC) with the TLR2 mutation Arg677Trp, a recently reported polymorphism that is associated with lepromatous leprosy. In leprosy patients with the TLR2 mutation, production of IL-2, IL-12, IFN-γ, and TNF-α by M. leprae-stimulated PBMC were significantly decreased compared with that in groups with wild-type TLR2. However, the cells from patients with the TLR2 mutation showed significantly increased production of IL-10. There was no significant difference in IL-4 production between the mutant and wild-type during stimulation. Thus, these results suggest that the TLR2 signal pathway plays a critical role in the alteration of cytokine profiles in PBMC from leprosy patients and the TLR2 mutation Arg677Trp provides a mechanism for the poor cellular immune response associated with lepromatous leprosy.     

Enhancement of toll-like receptor 2-mediated immune responses by AIMP1, a novel cytokine, in mouse dendritic cells

Aminoacyl tRNA synthetase-interacting protein 1 (AIMP1) is a novel pleiotropic cytokine that was identified initially from Meth A-induced fibrosarcoma. It is expressed in the salivary glands, small intestine and large intestine, and is associated with the innate immune system. Previously, we demonstrated that AIMP1 might function as a regulator of innate immune responses by inducing the maturation and activation of bone-marrow-derived dendritic cells (BM-DCs). Toll-like receptors (TLRs) are major pathogen-recognition receptors that are constitutively expressed on DCs. In this study, we attempted to determine whether AIMP1 is capable of regulating the expression of TLRs, and also capable of affecting the TLR-mediated activation of DCs. Expression of TLR1, -2, -3 and -7 was highly induced by AIMP1 treatment in BM-DCs, whereas the expression of other TLRs was either down-regulated or remained unchanged. In particular, the expression of the TLR2 protein was up-regulated by AIMP1 in a time-dependent and dose-dependent manner, and was suppressed upon the addition of BAY11-7082, an inhibitor of nuclear factor-κB. AIMP1 was also shown to increase nuclear factor-κB binding activity. Importantly, AIMP1 enhanced the production of interleukin-6 and interleukin-12, and the expression of co-stimulatory molecules on BM-DCs when combined with lipoteichoic acid or Pam3Cys, two well-known TLR2 agonists. Collectively, these results demonstrate that the AIMP1 protein enhances TLR2-mediated immune responses via the up-regulation of TLR2 expression.     

Identification of two subpopulations of purified human blood B cells, CD27- CD23+ and CD27high CD80+, that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6

B‐cell expression of certain Toll‐like receptors (TLRs) is important in linking innate and adaptive immune responses in normal and pathological conditions. The expression of TLR9 plays a role in the recognition of conserved pathogen motifs in a manner that is dependent on B‐cell localization, deduced from B‐cell phenotype. The nature of TLR9 function is unclear. A first step in unravelling the function of this pattern recognition receptor is to discover the precise nature of the cell types that express TLR9. This study used three‐colour flow cytometry to characterize the B lymphocytes from human peripheral blood mononuclear cells (PBMCs) that express TLR9 on the surface. We sorted TLR9‐positive B and non‐B cells from the PBMC population and detected TLR9 expression on naïve and memory B cells. Moreover, we identified two discrete subpopulations of B cells: CD19⁺ CD27^? CD23⁺ cells and CD19⁺ CD27^high CD80⁺ cells. These subpopulations expressed high levels of membrane TLR9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of interleukin‐6 (compared to controls). Our finding that this pattern recognition receptor is expressed on a variety of cell subsets adds to the current understanding of the functional complexity of B‐cell membrane TLR9.     

Bruton's tyrosine kinase regulates TLR9 but not TLR7 signaling in human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) represent a key cell type for both innate and adaptive immunity. PDCs express both TLR7 and TLR9 and the recognition of nucleic acids by these two receptors triggers the production of a large amount of type‐I IFN and the induction of PDC maturation into APCs. This unique feature of PDCs is at the basis of clinical development of both TLR7 and TLR9 agonists for infectious diseases, allergy, cancer, and asthma. However, TLR7 and TLR9 recognition of self‐nucleic acids is linked to many autoimmune diseases including lupus, and a better understanding of the signaling pathways of these two receptors in PDCs is thus important. We have identified Bruton's tyrosine kinase (Btk) as an important player for TLR9 but not TLR7 signaling in human PDCs. Blocking Btk using a specific inhibitor leads to the reduction of all TLR9‐induced responses in PDCs, including cytokine production and expression of costimulatory molecules, while this has no impact on the TLR7 response. This identifies Btk as a key molecule in TLR9 signaling in PDCs and is the first demonstration that the TLR7 and TLR9 pathways can be dissociated in human PDCs.     

Distinct regulation of CD40-mediated interleukin-6 and interleukin-12 productions via mitogen-activated protein kinase and nuclear factor kappaB-inducing kinase in mature dendritic cells

The role of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) pathways, especially NF-kappaB-inducing kinase (NIK)-mediated alternative pathway, in CD40-mediated interleukin (IL)-6 and IL-12 productions by immature or mature dendritic cells (DCs) was investigated. Murine myeloid DCs were matured by treatment with lipopolysaccharide. CD40 ligation induced modest or vigorous cytokine productions in immature or mature DCs, respectively. After CD40 ligation, p38 MAPK was significantly activated in either immature or mature DCs. SB203580, a p38 MAPK inhibitor, markedly decreased CD40-mediated IL-6 and IL-12 productions in immature DCs. In mature DCs, SB203580 significantly decreased CD40-mediated IL-6 but not IL-12 production. On the other hand, CD40 ligation induced vigorous activation of the NF-kappaB alternative pathway including p100 phosphorylation and subsequent nuclear translocations of p52, a processed form of p100, and RelB in mature but not immature DCs. The CD40-mediated phosphorylation of p100 was completely abolished in NIK-mutated mature DCs. The NIK mutation markedly reduced CD40-mediated IL-12 but not IL-6 production by mature DCs. Taken together, we concluded that IL-6 and IL-12 productions in response to CD40 ligation were controlled by p38 MAPK and NIK mediated alternative pathway, respectively, in mature DCs.     

Human Langerhans' cells and dermal-type dendritic cells generated from CD34 stem cells express different toll-like receptors and secrete different cytokines in response to toll-like receptor ligands

Langerhans' cells (LC) and dermal dendritic cells (dDC) are located in the superficial and deeper layers of the skin respectively and represent the main dendritic cell (DC) populations of the skin. LC-like and dDC-like DC can be generated from CD34 stem cells and this system is widely used as a model for investigating these cells and in therapeutic vaccination. Here we report toll-like receptor (TLR) expression in human LC and dDC derived from CD34 stem cells. In vitro-generated DC expressed TLR-1, 2, 4, 6, 8 and 10. LC, but not dDC, expressed TLR-5, whereas only dDC expressed TLR-3. Maturation of LC was mediated by TLR-2, 4 and 5 ligands, but not by a TLR-3 ligand. dDC maturation was induced by TLR-3 and -4, but not with TLR-5 ligand and only weakly by a TLR-2 ligand. Stimulated LC secreted interleukin (IL)-1beta, low levels of tumour necrosis factor-alpha (TNF-alpha) and IL-8, but not IL-6 or IL-10. dDC secreted TNF-alpha, IL-6, IL-8 and IL-10, but little IL-1beta. IL-12p70 was not produced by ligand-stimulated dDC or LC, but was secreted by monocyte-derived DC (mdDC) stimulated with lipopolysaccharide (LPS). Thus, in vitro-generated LC and dDC detect different pathogen-associated molecules and show different cytokine-secretion profiles in response to TLR ligands.     

Murine thymic plasmacytoid dendritic cells

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c(+)B220(+) subset expresses Ly6C(high) and MHC class II(low) in contrast with previously described thymic DC (CD11c(+)B220(-) cells). Freshly isolated thymic CD11c(+)B220(+) cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN-alpha following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.     

Freeze-and-thaw-disrupted tumour cells impair the responsiveness of DC to TLR stimulation

Cancer immunotherapy aims at inducing immune responses against tumour-associated antigens that mediate the eradication of tumour cells. For successful vaccination against antigens expressed by the tumour, the immune system has to be provided with sufficient amounts of these antigens in connection with strong immunostimulatory signals such as toll-like receptor (TLR) ligands. Tumour cells represent a convenient source of relevant tumour-associated antigens but can have suppressive properties. In this study, we explored how different forms of tumour cell material influence the activation of dendritic cells (DC), which play a crucial role in the induction of anti-tumour immune responses. We show that freeze-and-thaw-disrupted tumour cells inhibit DC activation in response to TLR stimulation, a phenomenon that is only partially seen with non-disrupted control cells. This suppression of DC stimulation is independent of tumour cell- and species-specific factors. We tested the hypothesis that phosphatidylserine on cells with disrupted membrane integrity mediates inhibition of TLR-induced DC activation. Our experimental evidence indicates that phosphatidylserine is not involved in the inhibition of TLR-mediated DC activation by freeze-and-thaw-disrupted cells. The inhibitory activity associated with disrupted tumour cells could explain why such preparations are less effective tumour vaccines than apoptotic tumour cells.     

Altered innate immune response of plasmacytoid dendritic cells in multiple sclerosis

Plasmacytoid dendritic cells (pDCs) are of crucial importance in immune regulation and response to microbial factors. In multiple sclerosis (MS), pDCs from peripheral blood showed an immature phenotype, but its role in susceptibility to MS is not determined. Because infectious diseases are established triggers of exacerbations in MS, in this study we have characterized the expression of Toll‐like receptors (TLR) and the maturation and functional properties of peripheral blood pDCs from clinically stable, untreated MS patients in response to signals of innate immunity. After stimulation of TLR‐9, interferon (IFN)‐α production by pDCs was significantly lower in MS (n = 12) compared to healthy controls (n = 9). In an allogenic two‐step co‐culture assay we found an impaired effect of TLR‐9 stimulation on IFN‐γ expression of autologous naive T cells in MS patients (n = 4). In peripheral blood mononuclear cells, TLR‐9 stimulation with type A CpG ODN resulted in a higher expression of TLR‐1, ‐2, ‐4, ‐5 and ‐8 in MS patients (n = 7) compared with healthy controls (n = 11). These findings suggest an altered innate immune response to microbial stimuli in MS patients and may help understanding of why common infectious agents trigger MS attacks.     

Modification of TLR-induced activation of human dendritic cells by type I IFN: synergistic interaction with TLR4 but not TLR3 agonists

Upon detection of direct and indirect signs of infection, dendritic cells (DC) undergo functional changes that modify their ability to elicit immune responses. Type I interferon (IFN-alpha/beta), which includes a large family of closely related infection-inducible cytokines, represents one indirect signal that can act as a DC stimulus. We have investigated the ability of IFN-alpha/beta subtypes to affect DC function and to influence DC responses to Toll-like receptor (TLR) agonists (i.e., direct infection-associated signals). Subtle differences were observed among 15 subtypes of IFN-alpha/beta in the ability to stimulate expression of maturation markers and chemokines by human monocyte-derived DC, with IFN-omega being the most unique in its effects. Pre-treatment with IFN-alpha/beta did not alter the ability of DC to mature in response to subsequent contact with TLR agonists, but did modulate their secretion of chemokines. Conversely, IFN-alpha/beta was shown to act synergistically with TLR4 but not TLR3 agonists for the induction of maturation and chemokine production when DC were exposed to IFN-alpha/beta and TLR ligands simultaneously. Taken together, these results indicate a complex role for IFN-alpha/beta in regulating DC function during the course an infection, which varies according to IFN-alpha/beta subtype and the timing of exposure to other stimuli.     

DiC14-amidine cationic liposomes stimulate myeloid dendritic cells through Toll-like receptor 4

DiC14-amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14-amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14-amidine liposomes induced the production of IL-12p40 and TNF-alpha by mouse bone marrow-derived DC. DiC14-amidine liposomes also activated human DC, as shown by synthesis of IL-12p40 and TNF-alpha, accumulation of IL-6, IFN-beta and CXCL10 mRNA, and up-regulation of membrane expression of CD80 and CD86. DC stimulation by diC14-amidine liposomes was associated with activation of NF-kappaB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14-amidine liposomes use Toll-like receptor 4 to elicit both MyD88-dependent and Toll/IL-1R-containing adaptor inducing interferon IFN-beta (TRIF)-dependent responses.     

Role of natural interferon-producing cells and T lymphocytes in porcine monocyte-derived dendritic cell maturation

Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.