Aflatoxin B1 interferes with the antigen-presenting capacity of porcine dendritic cells

Aflatoxins (AFs) are harmful to animal and human health upon consumption of AF-contaminated feed or food. Among many forms of AFs, aflatoxin B₁ (AFB₁) is the most toxic and carcinogenic. In addition, AFB₁ impairs cell-mediated immunity, although the exact mechanism of this immunotoxicity is currently unknown. By far the most pivotal cells in the induction of immune responses are dendritic cells (DCs). These highly specialised cells dictate T-cell polarisation depending on the nature of the encountered antigens and environmental cues. To elucidate the effect of AFB₁ on the function of DCs, we used porcine monocyte-derived DCs (MoDCs) as a model system. A low dose of AFB₁ transiently reduced the phagocytic capacity of MoDCs. Furthermore, as compared to untreated MoDCs, AFB₁ significantly downregulated the cell surface expression of the co-stimulatory molecule CD40 at 12 h post treatment, while at 24 h the membrane expression levels of CD40 and the activation marker CD25 were significantly upregulated. Interestingly, the T-cell proliferation-inducing capacity of DCs was diminished upon AFB₁ treatment. In contrast, the cytokine secretion pattern of AFB₁-treated MoDCs was similar to mock-treated MoDCs. The results in this study indicate that a low level of AFB₁ dysregulates the antigen-presenting capacity of DCs, which could explain the observed immunotoxicity of this mycotoxin, and further stress the need to reduce AFB₁ levels in agricultural commodities.     

Loxoribine,a selective Toll-like receptor 7 agonist,induces maturation of human monocyte-derived dendritic cells and stimulates their Th-1- and Th-17-polarizing capability

Recently, a guanosine analog, 7-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), has been identified as a selective Toll-like receptor (TLR)7 agonist. Bearing in mind the controversy regarding the expression of TLR7 by human myeloid dendritic cells (DCs) and its significance for functions of these cells, the goal of this study was to investigate the effect of loxoribine on differentiation, maturation and functions of human monocyte-derived (Mo)DCs. Immature MoDCs were obtained by cultivation of monocytes for 6 days with recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. These cells were stimulated with loxoribine (250 μM) for an additional 48 h. Phenotypic properties of MoDCs were determined by flow cytometry, cytokine production was assayed by ELISA, whereas their allostimulatory capability was tested using a mixed leukocyte reaction. We showed that loxoribine up-regulated the expression of TLR7, CD40, CD54, CD80, CD83 and CCR7 and stimulated the production of IL-12, IL-23, IL-27 and IL-10 by MoDCs, whereas the level of interferon (IFN)-β was not modulated. Allogeneic CD4⁺T cells in co-culture with loxoribine-treated MoDCs proliferated more strongly, at lower DC/CD4⁺T-cell ratio (1:80), and secreted significantly higher levels of IL-17 and IFN-γ compared to the cultures with control MoDCs. The stimulatory effect of loxoribine on T helper (Th)1 polarization capability of MoDCs was further potentiated by ligation of CD40. In conclusion, our results show that loxoribine stimulated differentiation, maturation, allostimulatory as well as Th1 and Th17 polarization capability of human MoDCs and suggests that these effects might be associated with up-regulation of TLR7 expression, but not increased IFN-β production.     

Sensitization assays: monocyte-derived dendritic cells versus a monocytic cell line (THP-1)

Dendritic cells (DCs) play a critical role in the skin sensitization process of contact allergens. Many efforts have been made to develop in vitro sensitization tests that employ DCs, but more recently protocols were introduced that use cell lines other than DCs. The potential of the cell line THP-1 compared to monocyte-derived DCs (MoDCs) was evaluated using a known potent sensitizer 2,4-dinitrochlorobenzene (DNCB) and the terpenoid ascaridol (1,4-epodioxy-p-menth-2-ene), an ingredient present in oxidized tea tree oil. Activation of these cells was studied by estimation of the CD86 and CD54 cell surface expression. Overall, comparable results were found. The expression of CD86 was augmented by ascaridol in THP-1 and MoDCs, while the expression of CD54 was not reproducibly increased. These results encourage the further development of THP-1 cells as a short-term model for sensitization testing.     

Recombinant human intelectin binds bovine lactoferrin and its peptides

Intelectin (IntL), a lectin that exists on the brush border membrane of the small intestine, plays a role in the innate immune response and also acts as a receptor for lactoferrin (LF), an iron-binding glycoprotein found in milk and other secretions. Similar to human LF (hLF), bovine LF (bLF) has been shown to induce proliferation and differentiation of human enterocytes and to modulate their cytokine productions. To evaluate the interaction between human IntL (hIntL) and bLF, recombinant hIntL (rhIntL) conjugated with a tag sequence was examined for its ligand-binding capacity by using microtiter plates coated with LF or other proteins. Interestingly, rhIntL showed higher binding for bLF than hLF. It also bound pepsin hydrolysate of bLF, but to a lower degree than native bLF. A very low binding of rhIntL was observed for bovine serum albumin or transferrin. These findings suggest that hIntL acts as a receptor for bLF and its digested fragments.     

CCL21-induced immune cell infiltration

Cellular immune responses can be initiated via peptide presentation by specialized antigen presenting cells, dendritic cells (DCs), which stimulate naïve T cells. The trafficking of DCs and T cells is regulated by chemokines such as CCL21. CCL21 is normally expressed in the lymphoid organs and coordinates the interactions between DCs and T cells, thereby contributing to the initiation of T cell responses. In order to comprehend the mechanisms of CCL21 activity and to utilize CCL21 optimally in therapy, understanding the kinetics of the responses of various cell types to CCL21 would be beneficial. Therefore, in this study, we injected mice subcutaneously (s.c.) with CCL21 and examined the DC and T cell infiltration of the local draining lymph node. CCL21 injection resulted in significantly increased numbers of lymphoid and myeloid DCs and effector T lymphocytes in the local node at 4 days. Furthermore, at 4 days small lymphoid-like structures were visible in the injection areas. These results provide guidance for the optimal timing of CCL21 use in combination with vaccines.     

Mannose-functionalized "pathogen-like" polyanhydride nanoparticles target C-type lectin receptors on dendritic cells

Targeting pathogen recognition receptors on dendritic cells (DCs) offers the advantage of triggering specific signaling pathways to induce a tailored and robust immune response. In this work, we describe a novel approach to targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide "pathogen-like" properties that ensure nanoparticles engage C-type lectin receptors on DCs. The surface of polyanhydride nanoparticles was functionalized by covalent linkage of dimannose and lactose residues using an amine-carboxylic acid coupling reaction. Coculture of functionalized nanoparticles with bone marrow-derived DCs significantly increased cell surface expression of MHC II, the T cell costimulatory molecules CD86 and CD40, the C-type lectin receptor CIRE and the mannose receptor CD206 over the nonfunctionalized nanoparticles. Both nonfunctionalized and functionalized nanoparticles were efficiently internalized by DCs, indicating that internalization of functionalized nanoparticles was necessary but not sufficient to activate DCs. Blocking the mannose and CIRE receptors prior to the addition of functionalized nanoparticles to the culture inhibited the increased surface expression of MHC II, CD40 and CD86. Together, these data indicate that engagement of CIRE and the mannose receptor is a key mechanism by which functionalized nanoparticles activate DCs. These studies provide valuable insights into the rational design of targeted nanovaccine platforms to induce robust immune responses and improve vaccine efficacy.     

Biochemical characterization of bovine lactoferrin as a glycyrrhizin-binding protein in vitro

Lactoferrin (LF) from bovine colostrum was biochemically characterized as a glycyrrhizin (GL)-binding protein (gbP) in vitro. It was found that (i) bovine LF (bLF) and a synthetic bovine lactoferricin (bLFcin, the N'-terminal region of bLF at the positions 17--41) had a high affinity to a GL-affinity column; (ii) approximately 1.8 moles of GL were bound to a molecule of bLF with a binding constant of approx. 1.20x10(4) M(-1) at pH 6.8; and (iii) GL, but not glycyrrhetinic acid (GA), induced a conformational change of bLF. In addition, the glucuronic acid moiety of the GL molecule was found to be responsible for binding to bLF, because (i) no binding of GA and two glucoses-GA (Glc-Glc-GA) to bLF was detected; and (ii) a synthetic fluorinated GL (GlcA-GlcF-GA) and mono-glucuronyl-GA (mono-GlcA-GA) were bound significantly to bLF. A similar binding of GL to human LF (hLF) was also observed under the same experimental conditions. Data provided here suggest that (i) bLF contains plural GL-binding sites; and (ii) the specific binding of GL to bLF may modulate the physiological activity of bLF in vivo.     

Endogenous lectins shape the function of dendritic cells and tailor adaptive immunity: mechanisms and biomedical applications

In spite of their central role in orchestrating immunity, dendritic cells (DCs) can also limit harmful reactions and promote immune tolerance by inducing T cell anergy or favoring the differentiation of T regulatory (T(reg)) cells. Several factors may influence the 'decision' of DCs to become immunogenic or tolerogenic including the nature of antigenic challenge, the engagement of selective pathogen recognition receptors (PRRs) and the balance of cytokines and growth factors. In addition, mounting evidence indicates a key role of endogenous lectins including C-type lectins, siglecs and galectins in shaping DC immunogenicity and tailoring adaptive immune responses, through recognition of specific 'glycan signatures' on invading pathogens or host cells. While galectins are in general secreted proteins that act in a paracrine or autocrine manner, all known siglecs and most C-type lectins are membrane-bound receptors that convey glycan-containing information into DC differentiation or maturation programs. Yet, some of the signaling pathways triggered by endogenous lectins converge in similar functional outcomes regardless of divergences in their structure, homology or glycan-binding specificity. To gain a more complete understanding on the role of protein-glycan interactions in DC biology, here we will integrate scattered information on these structurally-divergent but functionally-related lectins and their potential biomedical applications.     

Expression of N-acetyltransferase in monocyte-derived dendritic cells

Dendritic cells (DCs) are known to internalize, process, and present low-molecular-weight chemicals to T cells in the course of the sensitization and elicitation phase of allergic contact dermatitis. Thus, DCs may be involved in metabolic activation and detoxification of haptens and thereby influence the quantity of immunogens inducing sensitization. Recently, the cytochrome P-450 enzymes expressed in monocyte-derived dendritic cells (MoDCs) were characterized. In the present study, N-acetyltransferase 1 and 2 (NAT-1 and -2) mRNA expression and N-acetylation capacities of these cells were investigated. Monocytes from healthy donors were incubated with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 6 d and the resulting immature MoDCs were characterized by flow cytometry. Total RNA from MoDCs was isolated, reverse transcribed, and polymerase chain reaction (PCR) for NAT-1 and NAT-2 mRNA was performed. Data showed the presence of mRNA for NAT-1 (9 of 10 donors) and NAT-2 (8 of 10 donors) in these cells. NAT-1 enzyme activities were achieved through acetylation of para-aminobenzoic acid (PABA) by MoDC cell lysates and activities varied between 23.4 and 26.6 nmol/mg/min. In addition, complete cell acetylation of para-phenylenediamine (PPD), estimated via analysis of monoacetyl-PPD (MAPPD) and diacetyl-PPD (DAPPD) in cell culture supernatants, confirmed that in vitro generated MoDCs (4 of 6 donors) express metabolic active N-acetyltransferase (NAT-1). In the case of PPD, our results emphasize that N-acetylation status may influence the amounts of immunogens available for sensitization to PPD.     

Interactions between dendritic cells and epithelial cells in allergic disease

Dendritic cells (DCs) play a crucial role in the sensitisation process. Upon encounter with an allergen, DCs require interactions with other cells and factors for triggering a primary or secondary immune response. Epithelial cells (ECs) express features of accessory cells, such as expression of HLA-DR, co-stimulatory molecules, functional FcgammaR, molecules of the antigen-processing machinery, and display an ability to internalise antigen. These features may authorize them to function as immunomodulators (e.g. amplification of memory T cells during secondary immune responses). ECs may increase chemokine (e.g. CCL20) secretion thereby attracting DCs. Epithelial human TSLP activates DC, which allow them to prime naive T cells for the production of pro-inflammatory cytokines, while down-regulating IFN-gamma and IL-10. ECs may also influence the local polarization of types l and 2 antigen-presenting cells via PGE(2) by impairing the ability of maturing DC to produce bioactive IL-12 p70. PGE(2) is synergistic with IL-1beta and TNF-alpha in the induction of functional and phenotypic maturation of DC and induce IL12 p40 production. Sensitisation via the respiratory route may be Th(2) skewed, possibly because the antigen recognition by DC occurs in an environment rich of airway EC-product such as PGE(2).     

Maturation-dependent expression of C1q binding proteins on the cell surface of human monocyte-derived dendritic cells

The expression and cell surface levels of many important receptors are dependent on the maturation stage of dendritic cells (DCs), and related to the unique function of immature and mature DCs. In this report, we show, for the first time, that human monocyte-derived DCs express two types of C1q receptors, gC1qR and cC1qR. Furthermore, immature DCs secrete detectable amount of C1q into the culture supernatant. Immature DCs express higher cell surface levels of both C1qRs than mature ones, while the total C1qR protein and mRNA levels remain the same. The following experimental evidence support this conclusion: (1) Inflammatory cytokines and LPS, which induce maturation of DCs, downregulate surface expression of both C1qR molecules. (2) Cytokines and drugs (IL-10, IFN-alpha, Dexamethasone), which keep DCs phenotypically and functionally immature, significantly upregulate the cell surface expression of both C1qRs. (3) Neither of these treatments changed the intracellular gC1qR level nor the gC1qR mRNA levels measured by real time RT-PCR. The elevated surface expression of C1qRs on DCs has been found to be not due to increased apoptosis or cell death as the result of DC treatment. Taken together, these data show that human monocyte-derived DCs express gC1qR and cC1qR, their expression on the cell surface is maturation dependent, and immature DCs secrete C1q. These data strongly suggest the role of C1qRs in immature DC function and in the regulation of immune processes.     

Antiviral activity of lactoferrin against canine herpesvirus

Lactoferrin (LF) is an iron-binding protein that is found in milk and other mammalian secretions. We found that bovine lactoferrin (bLF) inhibited both the in vitro infection and replication of canine herpesvirus (CHV) in Madin-Darby canine kidney (MDCK) cells. Incubation of CHV with bLF prevented subsequent infection of MDCK cells. Furthermore, proteins from CHV-infected MDCK cells were resolved by SDS–PAGE, and then bLF CHV-binding proteins were identified by far Western blotting. We demonstrated that the anti-CHV activity of bLF was due to its interaction with CHV as well as with MDCK cells. Both the apo- and holo-forms of bLF inhibited virus multiplication independently of their iron-withholding properties. We also demonstrated that human LF had anti-CHV activity. Our findings suggest that LF could be effective in dogs to provide protection against CHV infection.     

The active contribution of Toll-like receptors to allergic airway inflammation

Epithelia lining the respiratory tract represent a major portal of entry for microorganisms and allergens and are equipped with innate and adaptive immune signaling receptors for host protection. These include Toll-like receptors (TLRs) that recognize microbial components and evoke diverse responses in cells of the respiratory system. TLR stimulation by microorganism-derived molecules activates antigen presenting cells, control T helper (Th) 1, Th2, and Th17 immune cell differentiation, cytokine production by mast cells, and activation of eosinophils. It is clear that TLR are involved in the pathophysiology of allergic airway diseases such as asthma. Dendritic cells (DCs), a kind of antigen presenting cells, which play a key role in the induction of allergic airway inflammation, are privileged targets for pathogen associated molecular patterns (PAMPs). During the allergic responses, engagement of TLRs on DCs determines the Th2 polarization of the T cells. TLR signaling in mast cells increases the release of IL-5, and TLR activation of airway epithelial cells forces the generation of proallergic Th2 type of cytokines. Although these responses aim to protect the host, they may also result in inflammatory tissue damage in the airway. Under certain conditions, stimulation of TLRs, in particular, TLR9, may reduce Th2-dependent allergic inflammation by induction of Th1 responses. Therefore, understanding the complex regulatory roles of TLRs in the pathogenesis of allergic airway inflammation should facilitate the development of preventive and therapeutic measures for asthmatic patients.     

Pleurotus citrinopileatus polysaccharide induces activation of human dendritic cells through multiple pathways

Many edible mushrooms have become attractive as “health foods” and as source materials for immunomodulators. To increase our insight in the immune-modulatory properties of a polysaccharide of the oyster mushroom Pleurotus citrinopileatus, PCPS, we analyzed its effects on the function of human dendritic cells (DCs). We showed that PCPS induces upregulation of the surface maturation markers CD80, CD86 and HLA-DR on DCs, indicating its potential to induce DC maturation. In addition, PCPS stimulates DCs to secrete the pro-inflammatory cytokines TNF, IL-1β, IL-6 and IL-12, as well as the anti-inflammatory cytokine IL-10, and induces enhanced mRNA levels of the chemokines CCL2, CCL3, CCL8, CXCL9, CXCL10, and LTA. The secretion of TNF and IL-12 by PCPS-activated DCs could significantly be decreased by an anti-Dectin-1 antibody, as well as by a Syk kinase and a Raf-1 inhibitor, indicating that PCPS induces Dectin-1 signaling at least partly through the Syk- and the Raf-1-dependent pathways in DCs. Structural analysis of PCPS suggests that this polysaccharide is a β-1,3-branched β-1,6-glucan, which is in line with its capacity to activate Dectin-1. We showed that PCPS can induce TLR2 and TLR4, but not TLR3, signaling using TLR-HEK293 reporter cell lines. In human DCs, the effect of PCPS was additively increased by TLR4 activation, and synergistically enhanced by stimulation of TLR2, suggesting that interaction of PCPS with these TLRs contributes to the observed DC modulation. In conclusion, PCPS has the capacity to activate human DCs via multiple pathways.     

Maturation-dependent expression of C1q-binding proteins on the cell surface of human monocyte-derived dendritic cells

The expression and cell surface levels of many important receptors are dependent on the maturation stage of dendritic cells (DCs), and related to the unique function of immature and mature DCs. In this report, we show for the first time that human monocyte-derived DCs express two types of C1q-receptors, gC1qR and cC1qR. Furthermore, immature DCs secrete detectable amount of C1q into the culture supernatant. Immature DCs express higher cell surface levels of both C1qRs than mature ones, while the total C1qR protein and mRNA levels remain the same. The following experimental evidence supports this conclusion. (1) Inflammatory cytokines and LPS, which induce maturation of DCs, downregulate surface expression of both C1qR molecules. (2) Cytokines and drugs (IL-10, IFNalpha, dexamethasone) that keep DCs phenotypically and functionally immature significantly upregulate the cell surface expression of both C1qRs. (3) Neither of these treatments changed the intracellular gC1qR level nor the gC1qR mRNA levels measured by real-time RT-PCR. The elevated surface expression of C1qRs on DCs has been found not to be due to increased apoptosis or cell death as the result of DC treatment. Taken together, these data show that human monocyte-derived DCs express gC1qR and cC1qR, their expression on the cell surface is maturation dependent and imature DCs secrete C1q. These data strongly suggest the role of C1qRs in immature DC function and in the regulation of immune processes.     

Receptor-mediated delivery of antigens to dendritic cells: anticancer applications

Recently, there has been a surge of interest in the use of ex vivo antigen-pulsed dendritic cells (DCs) in the immunotherapy for cancer. DCs are powerful adjuvants with the ability to prime naive CD4+ and CD8+ T cells. As antigen sources, various preparations, including peptides, proteins, crude tumor lysates, and DCs transfected or transformed with various viruses, have been used. These procedures that involve the isolation of patient DCs and reintroduction after in vitro manipulation are time-consuming and expensive. The DC populations used frequently in ex vivo clinical studies are IL-4 and GM-CSF cultured DCs that may not represent the in vivo DC populations. An attractive method of targeting in vivo DCs is to utilize various ligands or antibodies that bind discrete populations of DCs. These cell surface receptors will direct the antigen to different antigen processing pathways depending on the targeted receptor to induce cytotoxic T cell or T helper responses. This review will discuss the various approaches and receptors that have been used for antigen targeting that may eventually be translated to alternative DC-based immunotherapies.     

Co-Activation of Cultured Human Natural Killer Cells: Enhanced Function and Decreased Inhibition

ABSTRACT

Natural killer (NK) cells are important immune effector cells that protect the organism against viral infections and cancer. The cytotoxic activity of NK cells is induced by the engagement of a number of different activating surface receptors and controlled by inhibitory receptors to ensure self-tolerance. Resting NK cells need to be co-activated by involvement of at least two distinct activating receptors in order to induce their functional activity. However, in cultured NK cells, which have been expanded in cytokines such as interleukin (IL)-2, the engagement of a single activating receptor may be sufficient to induce their function. Data demonstrated that also cultured NK cells may be co-activated by involvement of certain combinations of activating receptors. This co-activation results in enhanced activation of Vav-1 and ERK signaling pathways and produces greater degranulation. In addition to enhanced functionality, co-activation makes NK cells more resistant to the effect of inhibitory receptors, thereby inducing more potent and efficient NK cell responses.     

Inducible Expression of Functional Mu Opioid Receptors in Murine Dendritic Cells

Opioids are known to exert direct effects on the immune system, and the expression of functional opioid receptors has been reported on several immune cell types. Dendritic cells (DCs) are important inducers and regulators of immune responses. In this study, we investigated whether murine dendritic cells express functional mu opioid receptors (MOR). RT-PCR analysis and double immunofluorescence staining revealed the expression of MOR in activated murine dendritic cells. We also studied the dynamic expression of MOR messenger RNA in murine dendritic cells in response to different Toll-like receptor ligands. Functionally, treatment of DCs with endomorphin 1 (EM1), a specific agonist of MOR, can inhibit the forskolin-induced formation of cyclic adenosine monophosphate level in activated DCs. Moreover, EM1 treatment resulted in less activation of p38 MAPK and more activation of ERK signaling in lipopolysaccharide-stimulated DCs. Consistently, treatment of DCs with EM1 altered cytokine production by increasing IL-10 and decreasing IL-12 and IL-23. Our results suggest that MOR is inducibly expressed on activated DCs and functionally mediates EM1-induced effects on DCs. Thus, dendritic cells might be involved in crosstalk between the neuroendocrine and the immune system.     

Part I: targeted particles for cancer immunotherapy

Dendritic cells (DCs) are the key antigen presenting cells that link innate and adaptive immunity. In the periphery, DCs capture antigens, process them and migrate into the regional lymph nodes where they could initiate antigen specific T cell immune responses. Immunotherapeutic strategies that aim to deliver tumor antigens specifically to DCs could not only boost anti-tumor immune responses but also could alleviate non-specific immune activation and/or unwanted side effects. Nano-sized particulate delivery systems are efficient modalities that can deliver tumor antigens to DCs in a targeted and specific manner. This review will provide general information on the rationale behind targeting antigens to DCs and the crucial role of DCs in initiating antigen specific T cell responses. Different strategies that have been employed in delivering antigens to DCs will be also discussed. A special emphasis will be put on specific targeting of cancer vaccine formulations to DC-specific receptors (e.g. CD11c, CD40, Fcγ, CCR6, pathogenic recognition receptors such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs)).