Bone Marrow-derived Myeloid Dendritic Cells Research Articles

Adipose-derived stem cell modulate tolerogenic dendritic cell-induced T cell regulation is correlated with activation of Notch-NFκB signaling

BackgroundAdipose-derived stem cells (ASCs) are recognized for their potential immunomodulatory properties. In the immune system, tolerogenic dendritic cells (DCs), characterized by an immature phenotype, play a crucial role in inducing regulatory T cells (Tregs) and promoting immune tolerance. Notch1 signaling has been identified as a key regulator in the development and function of DCs. However, the precise involvement of Notch1 pathway in ASC-mediated modulation of tolerogenic DCs and its impact on immune modulation remain to be fully elucidated. This study aims to investigate the interplay between ASCs and DCs, focusing the role of Notch1 signaling and downstream pathways in ASC-modulated tolerogenic DCs. MethodsRat bone marrow-derived myeloid DCs were directly co-cultured with ASCs to generate ASC-treated DCs (ASC-DCs). Notch signaling was inhibited using DAPT, while NFκB pathways were inhibited by NEMO binding domain peptide and si-NIK. Flow cytometry assessed DC phenotypes. Real-time quantitative PCR, Western blotting and immunofluorescence determined the expression of Notch1, Jagged1 and the p52/RelB complex in ASC- DCs. ResultsNotch1 and Jagged1 were highly expressed on both DCs and ASCs. ASC-DCs displayed significantly reduced levels of CD80, CD86 and MHC II compared to mature DCs. Inhibiting the Notch pathway with DAPT reversed the dedifferentiation effects. The percentage of induced CD25+/FOXP3+/CD4+ Tregs decreased when ASC-DCs were treated with DAPT (inhibition of the Notch pathway) and si-NIK (inhibition of the non-canonical NFκB pathway). ConclusionsASCs induce DC tolerogenicity by inhibiting maturation and promoting downstream Treg generation, involving the Notch and NFκB pathways. ASC-induced tolerogenic DCs can be a potential immunomodulatory tool for clinical application.

Cytohistological correlation of Langerhans cell histiocytosis of the spine in a young adult

Langerhans cell histiocytosis (LCH) is a rare multisystem disorder with a common histopathological finding of Langerhans-type cell proliferation mixed with a milieu of inflammatory cells. Bone involvement is a common presentation occurring in children and young adults. Recent studies using cell-specific gene expression profiling suggest that LCH arises from bone marrow-derived immature myeloid dendritic cells. We present a case of solitary LCH of cervical vertebrae in a young adult with pain and numbness in the shoulder diagnosed on fine-needle aspiration and confirmed on histopathology and immunohistochemistry. It was treated with tumor resection and stabilization of the vertebrae.

The Fusion Protein rFlaA:Betv1 Modulates DC Responses by a p38-MAPK and COX2-Dependent Secretion of PGE2 from Epithelial Cells.

Developing new adjuvants/vaccines and better understanding their mode-of-action is an important task. To specifically improve birch pollen allergy treatment, we designed a fusion protein consisting of major birch pollen allergen Betv1 conjugated to the TLR5-ligand flagellin (rFlaA:Betv1). This study investigates the immune-modulatory effects of rFlaA:Betv1 on airway epithelial cells. LA-4 mouse lung epithelial cells were stimulated with rFlaA:Betv1 in the presence/absence of various inhibitors with cytokine- and chemokine secretion quantified by ELISA and activation of intracellular signaling cascades demonstrated by Western blot (WB). Either LA-4 cells or LA-4-derived supernatants were co-cultured with BALB/c bone marrow-derived myeloid dendritic cells (mDCs). Compared to equimolar amounts of flagellin and Betv1 provided as a mixture, rFlaA:Betv1 induced higher secretion of IL-6 and the chemokines CCL2 and CCL20 from LA-4 cells and a pronounced MAPK- and NFκB-activation. Mechanistically, rFlaA:Betv1 was taken up more strongly and the induced cytokine production was inhibited by NFκB-inhibitors, while ERK- and p38-MAPK-inhibitors only suppressed IL-6 and CCL2 secretion. In co-cultures of LA-4 cells with mDCs, rFlaA:Betv1-stimulated LA-4 cells p38-MAPK- and COX2-dependently secreted PGE2, which modulated DC responses by suppressing pro-inflammatory IL-12 and TNF-α secretion. Taken together, these results contribute to our understanding of the mechanisms underlying the strong immune-modulatory effects of flagellin-containing fusion proteins.

Rare Genetic Disorders Affecting the Periodontal Supporting Tissues in Adolescence

In adolescents periodontal destruction may be the primary manifestation of an as yet unrecognized rare systemic disease, and it may be up to the periodontist to make the correct tentative diagnosis. Many genetic diseases that present with primary periodontal manifestations in adolescence affect immune function, sometimes with only mild or absent systemic features. They include periodontal Ehlers-Danlos syndrome (lack of attached gingiva, various connective tissue abnormalities), Papillon-Lefèvre syndrome (palmoplantar hyperkeratosis), and plasminogen deficiency (fibrin deposition within mucous membranes). Other immune disorders with severe periodontitis manifesting in adolescence are usually diagnosed in early childhood due to unmistakeable systemic features. They include Cohen syndrome (developmental disorder, truncal obesity, and microcephaly), Hermansky-Pudlak Syndrome (oculocutaneous albinism, bleeding diathesis, and other systemic manifestations), glycogen storage disease type 1b, and Chediak-Higashi syndrome (pyogenic infections, albinism, and neuropathy). The structural integrity of periodontal tissue is affected in genodermatoses such as Kindler syndrome, a type of epidermolysis bullosa. In primary hyperoxaluria, inflammatory periodontal destruction is associated with renal calculi. Breakdown of periodontal tissues independent of dental plaque biofilm-induced periodontitis is found in hypophosphatasia (highly variable skeletal hypomineralization) or isolated odontohypophosphatasia, hypophosphatemic rickets and primary hyperparathyroidism. Finally, alveolar osteolysis mimicking localized periodontitis may be due to neoplastic processes, e.g., in neurofibromatosis type 1 (typical skin features including café au lait macules and neurofibromas), Langerhans cell histiocytosis (locally destructive proliferation of bone marrow-derived immature myeloid dendritic cells), and Gorham-Stout disease (diffuse cystic angiomatosis of bone).

Crucial Role of Nucleic Acid Sensing via Endosomal Toll-Like Receptors for the Defense of Streptococcus pyogenes in vitro and in vivo.

Streptococcus pyogenes is a major human pathogen causing a variety of diseases ranging from common pharyngitis to life-threatening soft tissue infections and sepsis. Microbial nucleic acids, especially bacterial RNA, have recently been recognized as a major group of pathogen-associated molecular patterns (PAMPs) involved in the detection of Streptococcus pyogenes via endosomal Toll-like receptors (TLRs) in vitro. However, the individual contribution and cooperation between TLRs as well as cell-type and strain specific differences in dependency on nucleic acid detection during S. pyogenes infection in vitro have not been clarified in detail. Moreover, the role of particularly bacterial RNA for the defense of S. pyogenes infection in vivo remains poorly defined. In this study, we report that in all investigated innate immune cells involved in the resolution of bacterial infections, including murine macrophages, dendritic cells and neutrophils, recognition of S. pyogenes strain ATCC12344 is almost completely dependent on nucleic acid sensing via endosomal TLRs at lower MOIs, whereas at higher MOIs, detection via TLR2 plays an additional, yet redundant role. We further demonstrate that different S. pyogenes strains display a considerable inter-strain variability with respect to their nucleic acid dependent recognition. Moreover, TLR13-dependent recognition of S. pyogenes RNA is largely non-redundant in bone marrow-derived macrophages (BMDMs), but less relevant in neutrophils and bone marrow-derived myeloid dendritic cells (BMDCs) for the induction of an innate immune response in vitro. In vivo, we show that a loss of nucleic acid sensing blunts early recognition of S. pyogenes, leading to a reduced local containment of the bacterial infection with subsequent pronounced systemic inflammation at later time points. Thus, our results argue for a crucial role of nucleic acid sensing via endosomal TLRs in defense of S. pyogenes infection both in vitro and in vivo.

Human Bone Marrow-Derived Myeloid Dendritic Cells Show an Immature Transcriptional and Functional Profile Compared to Their Peripheral Blood Counterparts and Separate from Slan+ Non-Classical Monocytes.

The human bone marrow (BM) gives rise to all distinct blood cell lineages, including CD1c+ (cDC2) and CD141+ (cDC1) myeloid dendritic cells (DC) and monocytes. These cell subsets are also present in peripheral blood (PB) and lymphoid tissues. However, the difference between the BM and PB compartment in terms of differentiation state and immunological role of DC is not yet known. The BM may represent both a site for development as well as a possible effector site and so far, little is known in this light with respect to different DC subsets. Using genome-wide transcriptional profiling we found clear differences between the BM and PB compartment and a location-dependent clustering for cDC2 and cDC1 was demonstrated. DC subsets from BM clustered together and separate from the corresponding subsets from PB, which similarly formed a cluster. In BM, a common proliferating and immature differentiating state was observed for the two DC subsets, whereas DC from the PB showed a more immune-activated mature profile. In contrast, BM-derived slan+ non-classical monocytes were closely related to their PB counterparts and not to DC subsets, implying a homogenous prolife irrespective of anatomical localization. Additional functional tests confirmed these transcriptional findings. DC-like functions were prominently exhibited by PB DC. They surpassed BM DC in maturation capacity, cytokine production, and induction of CD4+ and CD8+ T cell proliferation. This first study on myeloid DC in healthy human BM offers new information on steady state DC biology and could potentially serve as a starting point for further research on these immune cells in healthy conditions as well as in diseases.

The Major Surface Glycoprotein of Pneumocystis murina Does Not Activate Dendritic Cells.

The major surface glycoprotein (Msg) is the most abundant surface protein among Pneumocystis species. Given that Msg is present on both the cyst and trophic forms of Pneumocystis and that dendritic cells play a critical role in initiating host immune responses, we undertook studies to examine activation of bone marrow-derived myeloid dendritic cells by Msg purified from Pneumocystis murina. Incubation of dendritic cells with Msg did not lead to increased expression of CD40, CD80, CD86, or major histocompatibility complex class II or to increased secretion of any of 10 cytokines. Microarray analysis identified very few differentially expressed genes. In contrast, lipopolysaccharide-activated dendritic cells had positive results of all of these assays. However, Msg did bind to mouse mannose macrophage receptor and human DC-SIGN, 2 C-type lectins expressed by dendritic cells that are important in recognition of pathogen-associated high-mannose glycoproteins. Deglycosylation of Msg demonstrated that this binding was dependent on glycosylation. These studies suggest that Pneumocystis has developed a mechanism to avoid activation of dendritic cells, potentially by the previously identified loss of genes that are responsible for the high level of protein mannosylation found in other fungi.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Roles of SOCS1 for establishment of mycobacterial infection

Abstract Background Suppressor of cytokine signaling (SOCS) 1 plays a crucial role in the negative regulation of JAK/STAT pathway. The SOCS1 is strongly upregulated by infection with several pathogens include M. bovis bacillus Calmette-Guérin (BCG). Since the function of SOCS1 induced by infection is not fully understood. In present study, we establish recombinant BCG which express a dominant-negative SOCS1 (rBCG-SOCS1DN) which acts as antagonist for SOCS1 signals. Methods C57BL/6, Rag1−/−, Nos2−/− and Rag1−/−Nos2−/− (DKO) mice were inoculated intratracheally with rBCGSOCS1DN or vector control rBCG (rBCG-pSO). Lungs and bronchoalveolar lavage fluids were harvested and analyzed. The mRNA and supernatants from bone marrow-derived myeloid dendritic cells (BMmDCs) infected with rBCG were harvested and nos2 gene expression and the production of nitric oxcide (NO) were measured. These cells were analized by electron microscopy. Results The amount of rBCG-SOCS1DN was significantly reduced by not only C57BL/6 but also Rag1−/− mice comparison with those of rBCG-pSO. The cytokine/chemokine profiles were not significant difference between rBCG-SOCS1DN and rBCG-pSO; however, nos2 gene expression and NO release from BMmDCs infected with rBCG-SOCS1DN were significantly higher than BMmDCs infected with rBCG-pSO. In Nos2−/− and DKO mice, there were no significant difference of bacterial burdens between rBCG-SOCS1DN and rBCG-pSO. Furthermore, the BMmDCs infected rBCG-SOCS1DN were necrotic damaged less than BMmDCs infected with rBCG-pSO. Conclusions These data suggest that BCG induce SOCS1 activation for evasion of microbicidal mechanism by nitric oxide from the innate immune responses.

ID: 205: A new RNA-based adjuvant enhances virus-specific vaccine responses by locally activating DC and inducing IFN-I responses TLR- and RLH-dependently

Among new adjuvants conferring a TH1-shift, RNAdjuvant® consisting of non-coding RNA complexed with a cationic carrier peptide is a promising candidate. Upon RNAdjuvant® treatment, bone marrow-derived myeloid dendritic cells (BM-mDC) and plasmacytoid DC (BM-pDC) showed Toll-like receptor (TLR)7-dependent IFN-I induction and upregulation of CD69 and CD86. This result was verified by the ex vivo analysis of splenic DC. Immunization of mice with seasonal influenza vaccines plus RNAdjuvant® significantly enhanced vaccine-induced influenza-specific IgG responses. In particular, IgG2b and IgG2c antibody subclasses were elevated, indicating a TH1-biased immune response. RNAdjuvant® mediated enhancement of antibody responses was conferred by concomitant TLR- and RIG-I-like helicase (RLH)-dependent signaling. Thus, upon administration of RNAdjuvant® two different signaling pathways were induced, explaining the strong immunoenhancing effect of RNAdjuvant®. Notably, upon intramuscular (i.m.) injection of RNAdjuvant® IFN-I induction and CD69 upregulation on DC were only detectable in draining, but not in non-draining lymph nodes. Furthermore, transient lymphopenia as a sensitive readout of serum cytokine responses was only detected upon intravenous (i.v.), but not upon i.m., injection of RNAdjuvant®. These observations indicated that i.m. administration of RNAdjuvant® induced primarily local effects. In conclusion, RNAdjuvant® is a novel efficacious immune enhancer that augments vaccine-induced TH1 responses in a MyD88- and Cardif-dependent manner by triggering strictly local effects. Thus, RNAdjuvant® holds promise to be a particularly well tolerable new adjuvant also in humans.

Human adipose-derived mesenchymal stem cells engineered to secrete IL-10 inhibit APC function and limit CNS autoimmunity

Interleukin (IL)-10 is an important immunoregulatory cytokine shown to impact inflammatory processes as manifested in patients with multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). Several lines of evidence indicate that the effectiveness of IL-10-based therapies may be dependent on the timing and mode of delivery. In the present study we engineered the expression of IL-10 in human adipose-derived mesenchymal stem cells (Adi-IL-10-MSCs) and transplanted these cells early in the disease course to mice with EAE. Adi-IL-10-MSCs transplanted via the intraperitoneal route prevented or delayed the development of EAE. This protective effect was associated with several anti-inflammatory response mechanisms, including a reduction in peripheral T-cell proliferative responses, a decrease in pro-inflammatory cytokine secretion as well as a preferential inhibition of Th17-mediated neuroinflammation. In vitro analyses revealed that Adi-IL-10-MSCs inhibited the phenotypic maturation, cytokine production and antigen presenting capacity of bone marrow-derived myeloid dendritic cells, suggesting that the mechanism of action may involve an indirect effect on pathogenic T-cells via the modulation of antigen presenting cell function. Collectively, these results suggest that early intervention with gene modified Adi-MSCs may be beneficial for the treatment of autoimmune diseases such as MS.

사리장 처리에 의한 수지상세포의 성숙 유도

Dendritic cells (DCs) are professional antigen-presenting cells playing key roles in immune sentinels as initiators of T-cell responses against microbial pathogens and tumors. Sarijang, a folk sauce containing extracts of Rhynchosia nulubilis, Ulmus davidiana roots, Allium sativum, and Rhus Verniaiflura bark, has been used as a nonspecific immunostimulant for cancer patients. However, little is known about its immunomodulating effects or their mechanisms. In this study, we investigated whether sarijang induces phenotypic and functional maturation of DCs. For this study, murine bone marrow-derived myeloid DCs were cultured in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF), and the generated immature DCs were stimulated with sarijang or lipopolysaccharide (LPS). Our data indicated that sarijang significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) as well as major histocompatibility complex (MHC) II, as did LPS. The results provide new insight into the immunopharmacology of sarijang and suggest a novel approach to the manipulation of DC for therapeutic application.

Fusion protein of TLR5-ligand and allergen potentiates activation and IL-10 secretion in murine myeloid DC

Toll-like receptor ligands are immune-modulatory components linking innate and adaptive immune responses and are considered to be promising vaccine components. Objective of this study was to investigate the adjuvant activity of Listeria monocytogenesis-derived TLR5-ligand flagellin A (flaA) genetically fused to ovalbumin (Ova, major chicken white egg allergen) in a murine in vitro system. Recombinant flaA, rOva, and a fusion protein of rflaA and rOva (rflaA:Ova) were over-expressed in Escherchia coli and purified by FPLC. LPS depletion was confirmed by LAL test. TLR5-binding was evaluated by human and murine TLR5-transgenic HEK 293 cells. The immune-modulatory effect of rflaA:Ova and rflaA:Ova modified by reduction and alkylation on purified BALB/c bone marrow-derived myeloid (mDC) and plasmacytoid dendritic cells (pDC) was investigated by flow cytometry and intracellular cytokine staining (ICS). Dose-dependent IL-8 secretion from transgenic HEK 293 cells confirmed binding of rflaA and rflaA:Ova molecules to human and murine TLR5. Recombinant flaA showed similar biological reactivity to TLR5-ligand fliC derived from Salmonella typhimurium applied as positive control. Compared to rflaA, both rflaA:Ova preparations induced higher expression of maturation markers (CD40, CD69, CD80, and CD86) on mDC, whereas only CD69 and CD40 were upregulated on pDC. Moreover, IL-6 and IL-10 production by mDC was enhanced upon stimulation with rflaA:Ova constructs in comparison to an equimolar mixture of both proteins whereas pDC did not show secretion of the investigated cytokines. Any immunological effects of LPS can be excluded by depletion of endotoxins and the lack of IL-10 production upon proteinase K digestion of rflaA:Ova. In summary, the rflaA:Ova fusion proteins showed an enhanced immune modulating capacity in comparison to rflaA or the mixture of rflaA and antigen. Since the rflaA:Ova fusion proteins induce strong IL-10 induction they are considered as potential vaccine candidates to improve allergen-specific immunotherapy.

Cell-Specific Gene Expression in Langerhans Cell Histiocytosis Lesions Reveals a Distinct Profile Compared with Epidermal Langerhans Cells

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207(+) Langerhans cells (LCs) and lymphocytes that can arise in almost any tissue and cause significant morbidity and mortality. After decades of research, the cause of LCH remains speculative. A prevailing model suggests that LCH arises from malignant transformation and metastasis of epidermal LCs. In this study, CD207(+) cells and CD3(+) T cells were isolated from LCH lesions to determine cell-specific gene expression. Compared with control epidermal CD207(+) cells, the LCH CD207(+) cells yielded 2113 differentially expressed genes (false discovery rate < 0.01). Surprisingly, the expression of many genes previously associated with LCH, including cell-cycle regulators, proinflammatory cytokines, and chemokines, were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly overexpressed in LCH CD207(+) cells. Furthermore, several genes associated with immature myeloid dendritic cells were overexpressed in LCH CD207(+) cells. Compared with the peripheral CD3(+) cells from LCH patients, the LCH lesion CD3(+) cells yielded only 162 differentially regulated genes (false discovery rate < 0.01), and the expression profile of the LCH lesion CD3(+) cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4, and SPP1. Results from this study support a model of LCH pathogenesis in which lesions do not arise from epidermal LCs but from accumulation of bone marrow-derived immature myeloid dendritic cells that recruit activated lymphocytes.

Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: Role of macrophage scavenger receptor class A type I and II

The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods. It produces chemically glycated proteins termed advanced glycation end products (AGEs). The glycation structures of AGEs are suggested to function as pathogenesis-related immune epitopes in food allergy. This study aimed at defining the T-cell immunogenicity of food AGEs by using ovalbumin (OVA) as a model allergen. AGE-OVA was prepared by means of thermal processing of OVA in the presence of glucose. Activation of OVA-specific CD4(+) T cells by AGE-OVA was evaluated in cocultures with bone marrow-derived murine myeloid dendritic cells (mDCs) as antigen-presenting cells. The uptake mechanisms of mDCs for AGE-OVA were investigated by using inhibitors of putative cell-surface receptors for AGEs, as well as mDCs deficient for these receptors. Compared with the controls (native OVA and OVA thermally processed without glucose), AGE-OVA enhanced the activation of OVA-specific CD4(+) T cells on coculture with mDCs, indicating that the glycation of OVA enhanced the T-cell immunogenicity of the allergen. The mDC uptake of AGE-OVA was significantly higher than that of the controls. We identified scavenger receptor class A type I and II (SR-AI/II) as a mediator of the AGE-OVA uptake, whereas the receptor for AGEs and galectin-3 were not responsible. Importantly, the activation of OVA-specific CD4(+) T cells by AGE-OVA was attenuated on coculture with SR-AI/II-deficient mDCs. SR-AI/II targets AGE-OVA to the MHC class II loading pathway in mDCs, leading to an enhanced CD4(+) T-cell activation. The Maillard reaction might thus play an important role in the T-cell immunogenicity of food allergens.

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K, PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of I kappaB alpha. Hb-induced IDO expression was inhibited by inhibitors of PI3-kinase (PI3K), PKC and nuclear factor (NF)-kappaB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O(2) (-), H(2)O(2), and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-kappaB through the PI3K-PKC-ROS and PI3K-Akt pathways is required for the Hb-induced IDO expression in BMDCs.

Toll-Like Receptor 9-Dependent Activation of Myeloid Dendritic Cells by Deoxynucleic Acids fromCandida albicans

The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and beta-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Delta null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9(-/-) and MyD88(-/-) mice were used. In a luciferase reporter assay, NF-kappaB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9(-/-) mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif.

Distinct Patterns of Dendritic Cell Cytokine Release Stimulated by Fungal β-Glucans and Toll-Like Receptor Agonists

beta-Glucans derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Yeast glucan particles (YGPs) are highly purified Saccharomyces cerevisiae cell walls composed of beta1,6-branched beta1,3-d-glucan and free of mannans. YGPs stimulated secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in wild-type murine bone marrow-derived myeloid dendritic cells (BMDCs) but did not stimulate interleukin-12p70 (IL-12p70) production. A purified soluble beta1,6-branched beta1,3-d-glucan, scleroglucan, also stimulated TNF-alpha in BMDCs. These two beta-glucans failed to stimulate TNF-alpha in Dectin-1 (beta-glucan receptor) knockout BMDCs. Costimulation of wild-type BMDCs with beta-glucans and specific Toll-like receptor (TLR) ligands resulted in greatly enhanced TNF-alpha production but decreased IL-12p70 production compared with TLR agonists alone. The upregulation of TNF-alpha and downregulation of IL-12p70 required Dectin-1, but not IL-10. Gamma interferon (IFN-gamma) priming did not overcome IL-12p70 reduction by beta-glucans. Similar patterns of cytokine regulation were observed in human monocyte-derived dendritic cells (DCs) costimulated with YGPs and the TLR4 ligand lipopolysaccharide. Finally, costimulation of BMDCs with YGPs and either the TLR9 ligand, CpG, or the TLR2/1 ligand, Pam(3)CSK(4), resulted in upregulated secretion of IL-1alpha and IL-10 and downregulated secretion of IL-1beta, IL-6, and IFN-gamma-inducible protein 10 but had no significant effects on IL-12p40, keratinocyte-derived chemokine, monocyte chemotactic protein 1, or macrophage inflammatory protein alpha, compared with the TLR ligand alone. Thus, beta-glucans have distinct effects on cytokine responses following DC stimulation with different TLR agonists. These patterns of response might contribute to the skewing of immune responses during mycotic infections and have implications for the design of immunomodulators and vaccines containing beta-glucans.

Toll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidin of Staphylococcus aureus

Leukocidin (Luk), an exotoxin of Staphylococcus aureus consisting of LukF and LukS, is a hetero-oligomeric pore-forming cytolytic toxin toward human and rabbit polymorphonuclear leukocytes. However, it is uncertain how Luk affects the host immune response. In the present study, we investigated whether Luk has the ability to stimulate mouse bone marrow-derived myeloid dendritic cells (BM-DCs). LukF activated BM-DCs to generate IL-12p40 mRNA, induce intracellular expression and extracellular secretion of this cytokine and express CD40 on their surface, whereas LukS showed a much lower or marginal ability in the activation of BM-DCs than its counterpart component. Similarly, TNF-α was secreted by BM-DCs upon stimulation with these components. Combined addition of these components did not lead to a further increase in IL-12p40 secretion. IL-12p40 production caused by LukF was completely abrogated in BM-DCs from TLR4-deficient mice similarly to the response to lipopolysaccharide (LPS). Polymixin B did not affect the LukF-induced IL-12p40 production, although the same treatment completely inhibited the LPS-induced response. Boiling significantly inhibited the response caused by LukF, but not by LPS. Finally, in a luciferase reporter assay, LukF induced the activation of NF-κB in HEK293T cells transfected with TLR4, MD2 and CD14, whereas LukS did not show such activity. These results demonstrate that LukF caused the activation of BM-DCs by triggering a TLR4-dependent signaling pathway and suggests that Luk may affect the host inflammatory response as well as show a cytolytic effect on leukocytes.

Cerebrospinal Fluid Dendritic Cells Infiltrate the Brain Parenchyma and Target the Cervical Lymph Nodes under Neuroinflammatory Conditions

BackgroundIn many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions.Methodology/Principal FindingsTo address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres+ APCs or CFSE+ DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.Conclusions/SignificanceAltogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.