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.     

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.     

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.     

CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells

CD45 is known to regulate signalling through many different surface receptors in diverse haemopoietic cell types. Here we report for the first time that CD45-/- bone marrow dendritic cells (BMDC) are more activated than CD45+/+ cells and that tumour necrosis factor (TNF) and interleukin-6 (IL-6) production by BMDC and splenic dendritic cells (sDC), is increased following stimulation via Toll-like receptor (TLR)3 and TLR9. Nuclear factor-kappaB activation, an important downstream consequence of TLR3 and TLR9 signalling, is also increased in CD45-/- BMDC. BMDC of CD45-/- mice also produce more TNF and IL-6 following stimulation with the cytokines TNF and interferon-alpha. These results show that TLR signalling is increased in CD45-/- dendritic cells and imply that CD45 is a negative regulator of TLR and cytokine receptor signalling in dendritic cells.     

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.     

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.     

Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll‐like receptors (TLRs) and C‐type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c⁺, CD141⁺ and CD16⁺ myeloid DCs and CD123⁺ plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR‐directed targeting. Whole blood was obtained from 13 allergic and six non‐allergic individuals. Mononuclear cells were purified and multi‐colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141⁺ DCs from allergic compared with non‐allergic donors. The subset‐specific PRR expression profiles suggests individual responsiveness to PRR‐targeting and supports functional specialization.     

Protease‐activated receptor 2 signalling promotes dendritic cell antigen transport and T‐cell activation in vivo

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c^high DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44⁺ CD62⁻) CD4⁺ and CD8⁺ T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC⁺ DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0·95% versus 0·47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.     

Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection

Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis‐dependent manner. Compared with autophagy‐deficient Beclin‐1^+/? mice, we found that bone‐marrow‐derived DCs from wild‐type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild‐type BMDCs secreted higher levels of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐α), interferon‐β (IFN‐β), IL‐12p70 and IFN‐γ than did Beclin‐1^+/? BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs exhibited increased activation of extracellular signal‐regulated kinase, Jun N‐terminal kinase, p38, and nuclear factor‐κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co‐localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL‐6, TNF‐α and IFN‐β in H1N1‐infected BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs were more efficient in inducing allogeneic CD4⁺ T‐cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4⁺ Foxp3⁺ regulatory T‐cell differentiation. Moreover, WT BMDCs were more efficient at cross‐presenting the ovalbumin antigen to CD8⁺ T cells. We consistently found that Beclin‐1^+/? BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1‐specific CD4⁺ and CD8⁺ T‐cell responses, which produced lower levels of IL‐6, TNF‐α and IFN‐β in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.     

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (T_reg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (−64%) but TLR-9-induced IFN-α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4⁺forkhead box protein 3 (FoxP3)⁺ regulatory T cells, but did not affect the generation of suppressive CD8⁺CD38⁺lymphocyte activation gene (LAG)-3⁺ T_reg. In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4⁺ T cell proliferation and induce CD4⁺FoxP3⁺ regulatory T cell generation.     

Polysaccharide Agaricus blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to M1 type,which mediates inhibition of tumour immune‐evasion via the Toll‐like receptor 2 pathway

Gr‐1⁺ CD11b⁺ myeloid‐derived suppressor cells (MDSCs) accumulate in tumor‐bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti‐cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin‐6 (IL‐6), IL‐12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr‐1⁺ CD11b⁺ monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll‐like receptor 2 (TLR2) signal of Gr‐1⁺ CD11b⁺ MDSCs and increased their M1‐type macrophage characteristics, such as producing IL‐12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr‐1⁺ CD11b⁺ MDSCs by pAbM treatment had less ability to convert the CD4⁺ CD25^? cells into CD4⁺ CD25⁺ phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti‐tumour effect of pAbM was dependent on Gr‐1⁺ CD11b⁺ monocytes, nether CD8⁺ T cells nor CD4⁺ T cells. In addition to, pAbM did not inhibit tumour growth in TLR2^–/– mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti‐tumour effects through the opposite of the suppressive function of Gr‐1⁺ CD11b⁺ MDSCs.     

The pruritogenic mediator endothelin‐1 shifts the dendritic cell–T‐cell response toward Th17/Th1 polarization

Endothelin‐1 (ET‐1) is associated with skin diseases such as atopic dermatitis (AD) and psoriasis. ET‐1 is enhanced in the skin of patients AD and psoriasis. In addition, plasma levels of ET‐1 are elevated in AD and psoriasis. Although both AD and psoriasis are T‐cell–mediated skin diseases, the association between ET‐1 and the T‐cell immune response has not been clarified. To evaluate the role of ET‐1 in inflammatory skin disease, we sought to investigate the effects of ET‐1 on the functions of dendritic cells (DCs) and subsequent immune responses. For this purpose, we immunohistochemically confirmed the upregulation of ET‐1 in the epidermis of patients with AD or psoriasis. ET‐1 directly induced phenotypic maturation of bone marrow‐derived DCs (BMDCs). In addition, ET‐1 augmented the production of several cytokines and allogeneic stimulatory capacity of BMDCs. Interestingly, ET‐1–activated BMDCs primed T cells to produce Th1 and Th17 cytokines, but not Th2 cytokines. These findings indicate that ET‐1 polarizes the DC–T‐cell response toward Th17/1 differentiation and may augment the persistent course of inflammatory skin diseases.     

Uptake of the yeast Malassezia furfur and its allergenic components by human immature CD1a+ dendritic cells

Atopic dermatitis (AD) is a chronic inflammatory skin disease with increasing prevalence, though still little is known of the pathomechanisms and the causes of the disease. Patients with AD often have specific IgE reactivity to the yeast Malassezia furfur (M. furfur), present in the normal microflora on human skin. To investigate the possible interaction of immature and mature antigen-presenting dendritic cells with the yeast M. furfur and its allergenic components. Monocyte-derived dendritic cells (MDDCs) generated from human peripheral blood were allowed to interact with FITC-labelled whole M. furfur yeast cells, M. furfur extract, a recombinant allergen from M. furfur designated rMal f 5 and M. furfur mannan, in the absence of IgE antibodies. Interaction and uptake were detected using flow cytometry and confocal laser scanning microscopy. Internalization of M. furfur yeast cells and yeast components by immature MDDCs was found using confocal laser scanning microscopy. Results from flow cytometric studies showed that a median of 94% (range, 65-98%) of the immature CD1a+ MDDCs were M. furfur extract positive, 81% (75-97%) rMal f 5 positive and 93% (62-98%) mannan positive. Mature CD1a+ MDDCs were significantly less efficient in this respect, with the corresponding figures only 26% (6-37%, P < 0.01), 6% (2-15%, P < 0.05) and 32% (9-50%, P < 0.01), respectively. Uptake of the non-glycosylated rMal f 5 by immature CD1a+ MDDCs was decreased to 27% (15-38%) by inhibition of pinocytosis. The binding of M. furfur extract and mannan was inhibited in a dose-dependent manner by methyl-alpha-D-mannopyranoside, suggesting uptake via the mannose receptor. Human immature CD1a+ MDDCs can efficiently take up M. furfur and allergenic components from the yeast in the absence of IgE antibodies, implying that sensitization of AD patients to M. furfur can be mediated by immature dendritic cells in the skin.     

Patency of Litomosoides sigmodontis infection depends on Toll‐like receptor 4 whereas Toll‐like receptor 2 signalling influences filarial‐specific CD4+ T‐cell responses

BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life‐stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial‐specific profiles, this study compared differences in immune responses between Mf⁺ and Mf^– infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf⁺ mice produce significantly more interleukin‐5 (IL‐5) to filarial antigens but equal levels of IL‐10 when compared with Mf^– mice. However, isolated CD4⁺ T cells from Mf⁺ mice produced significantly higher amounts of all measured cytokines, including IL‐10, when compared with CD4⁺ T‐cell responses from Mf^– mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll‐like receptor‐2‐deficient (TLR2^?/?) and TLR4^?/? BALB/c mice. Ninety‐three per cent of L. sigmodontis‐exposed TLR4^?/? BALB/c mice became patent (Mf⁺) although worm numbers remained comparable to those in Mf⁺ wild‐type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf⁺ mice had significantly lower numbers of Foxp3⁺ regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4⁺ T cells from infected wild‐type mice with granulocyte–macrophage colony‐stimulating factor‐derived TLR2^?/? dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4⁺ T‐cell responses, respectively.     

Human and rhesus plasmacytoid dendritic cell and B-cell responses to Toll-like receptor stimulation

Interferon-α (IFN-α) produced at high levels by human plasmacytoid dendritic cells (pDCs) can specifically regulate B-cell activation to Toll-like receptor (TLR) 7/8 stimulation. To explore the influence of IFN-α and pDCs on B-cell functions in vivo, studies in non-human primates that closely resemble humans in terms of TLR expression on different subsets of immune cells are valuable. Here, we performed a side-by side comparison of the response pattern between human and rhesus macaque B cells and pDCs in vitro to well-defined TLR ligands and tested whether IFN-α enhanced B-cell function comparably. We found that both human and rhesus B cells proliferated while pDCs from both species produced high levels of IFN-α in response to ligands targeting TLR7/8 and TLR9. Both human and rhesus B-cell proliferation to TLR7/8 ligand and CpG class C was significantly increased in the presence of IFN-α. Although both human and rhesus B cells produced IgM upon stimulation, only human B cells acquired high expression of CD27 associated with plasmablast formation. Instead, rhesus B-cell differentiation and IgM levels correlated to down-regulation of CD20. These data suggest that the response pattern of human and rhesus B cells and pDCs to TLR7/8 and TLR9 is similar, although some differences in the cell surface phenotype of the differentiating cells exist. A more thorough understanding of potential similarities and differences between human and rhesus cells and their response to potential vaccine components will provide important information for translating non-human primate studies into human trials.     

The effect of human immunodeficiency virus-1 on monocyte-derived dendritic cell maturation and function

Dendritic cells (DC) are mediators of the adaptive immune response responsible for antigen presentation to naive T cells in secondary lymph organs. Human immunodeficiency virus (HIV-1) has been reported to inhibit the maturation of DC, but a clear link between maturation and function has not been elucidated. To understand further the effects of HIV-1 on DC maturation and function, we expanded upon previous investigations and assessed the effects of HIV-1 infection on the expression of surface molecules, carbohydrate endocytosis, antigen presentation and lipopolysaccharide (LPS) responsiveness over the course of maturation. In vitro infection with HIV-1 resulted in an increase in the expression of DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as well as decreases in maturation-induced CCR7 and major histocompatibility complex (MHC)-II expression. Retention of endocytosis that normally occurs with DC maturation as well as inhibition of antigen presentation to CD8(+) T cells was also observed. Mitogen-activated protein kinase (MAPK) responsiveness to LPS as measured by phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK)1/2 was not affected by HIV-1 infection. In summary, in-vitro HIV-1 impairs DC maturation, as defined by cell surface protein expression, with selective alterations in mature DC function. Understanding the mechanisms of DC dysfunction in HIV infection will provide further insight into HIV immune pathogenesis.     

The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells

Dendritic cells (DC) play a key role in anti-viral immunity. Direct interactions between DC and hepatitis B virus (HBV) may explain the impaired DC function and the ineffective anti-viral response of chronic HBV patients resulting in HBV persistence. Here, the interaction between HBV surface antigens (HBsAg) and DC and the receptor involved were examined by flow cytometry in blood and liver tissue of HBV patients. The in vitro data showed that the mannose receptor (MR) is involved in HBsAg recognition and uptake by DC. The presence of HBsAg-positive DC was demonstrated sporadically in blood, but frequently in the liver of HBV patients. Interestingly, a positive correlation was found between HBsAg positivity and MR expression level in both liver- and blood-derived DC. These data suggest that in HBV infected patients, MR-mediated interaction between HBsAg and DC and subsequent impairment of DC predominantly occurs at the main site of infection, the liver.     

Sequence independent interferon‐α induction by multimerized phosphodiester DNA depends on spatial regulation of Toll‐like receptor‐9 activation in plasmacytoid dendritic cells

Single‐stranded versus multimeric phosphorothioate‐modified CpG oligodeoxynucleotides (ODNs) undergo differential endosomal trafficking upon uptake into plasmacytoid dendritic cells (pDCs), correlating with Toll‐like receptor‐9‐dependent pDC maturation/activation (single‐stranded B‐type CpG ODN) or interferon‐α (IFN‐α) induction (multimeric A‐type CpG ODN), respectively. As was recently shown, IFN‐α production, other than by CpG ODNs, can also be induced in a sequence‐independent manner by phosphodiester (PD) ODNs multimerized by 3′ poly‐guanosine (poly‐G) tails. We investigate here the type of endosomal trafficking responsible for IFN‐α induction by natural PD ODN ligands. We show that 3′ extension with poly‐G tails leads to multimerization of single‐stranded PD ODNs and to enhanced cellular uptake into pDCs. While monomeric PD ODNs, which induce CpG‐dependent Toll‐like receptor‐9 stimulation and pDC maturation/activation, localized to late endosomal/lysosomal compartments, the poly‐G multimerized PD ODNs, which induce CpG‐independent IFN‐α production, located to vesicles with a distinct, ‘early’ endosomal phenotype. We conclude that poly‐G‐mediated multimerization of natural PD ODNs allows for sequence‐independent, Toll‐like receptor‐9‐dependent IFN‐α induction in pDCs by combining three distinct effects: relative protection of sensitive PD ODNs from extracellular and intracellular DNase degradation, enhanced cellular uptake and preferential early endosomal compartmentation.