Kitti Pazmandi

Pollen-Induced Oxidative Stress Influences Both Innate and Adaptive Immune Responses via Altering Dendritic Cell Functions

It has been demonstrated that pollen grains contain NAD(P)H oxidases that induce oxidative stress in the airways, and this oxidative insult is critical for the development of allergic inflammation in sensitized mice. On the basis of this observation, we have examined whether pollen grain exposure triggers oxidative stress in dendritic cells (DCs), altering their functions. To test this hypothesis, human monocyte-derived DCs were treated with ragweed pollen grains. Our findings show that exposure to pollen grains induces an increase in the intracellular levels of reactive oxygen species in DCs. Our data also indicate that besides the NAD(P)H oxidases, other component(s) of pollen grains contributes to this phenomenon. Elevated levels of intracellular reactive oxygen species triggered the production of IL-8 as well as proinflammatory cytokines, such as TNF-α and IL-6. Treatment with pollen grains initiated the maturation of DCs, strongly upregulated the membrane expression of CD80, CD86, CD83, and HLA-DR, and caused only a slight increase in the expression of CD40. The pollen-treated DCs induced the development of naive T lymphocytes toward effector T cells with a mixed profile of cytokine production. Antioxidant inhibited both the phenotypic and functional changes of DCs, underlining the importance of oxidative stress in these processes. Collectively, these data show that pollen exposure-induced oxidative stress may contribute to local innate immunity and participate in the initiation of adaptive immune responses to pollen Ags.

Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells

Inflammation is associated with oxidative stress and characterized by elevated levels of damage-associated molecular pattern (DAMP) molecules released from injured or even living cells into the surrounding microenvironment. One of these endogenous danger signals is the extracellular mitochondrial DNA (mtDNA) containing evolutionary conserved unmethylated CpG repeats. Increased levels of reactive oxygen species (ROS) generated by recruited inflammatory cells modify mtDNA oxidatively, resulting primarily in accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) lesions. In this study, we examined the impact of native and oxidatively modified mtDNAs on the phenotypic and functional properties of plasmacytoid dendritic cells (pDCs), which possess a fundamental role in the regulation of inflammation and T cell immunity. Treatment of human primary pDCs with native mtDNA up-regulated the expression of a costimulatory molecule (CD86), a specific maturation marker (CD83), and a main antigen-presenting molecule (HLA-DQ) on the cell surface, as well as increased TNF-α and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-α secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide. Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-α production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs.

TLR ligands upregulate RIG-I expression in human plasmacytoid dendritic cells in a type I IFN-independent manner

Plasmacytoid dendritic cells (pDCs) are professional type I interferon (IFN)‐producing cells that play an essential role in antiviral immunity. In many cell types, detection of intracellular pathogens is mostly dependent on endosomal Toll‐like receptors (TLRs) and cytosolic sensors, such as retinoic acid‐inducible gene I (RIG‐I). However, the possible interplay between these two systems has not yet been elucidated. Here we aimed to study the collaboration of endosomal TLRs and RIG‐I in primary human pDCs. We found that under steady‐state conditions, pDCs express RIG‐I at very low level, but the expression of this receptor is rapidly and dramatically upregulated upon stimulation by the TLR7 ligand imiquimod or the TLR9 ligand type A CpG. We also demonstrated that pDCs are able to sense and respond to 5′‐triphosphate double‐stranded RNA (5′‐ppp‐dsRNA) only following activation by endosomal TLRs. Experiments on primary pDCs with functionally blocked IFN‐α/β receptor 1 (IFNAR1) and those on human pDC leukemia (pDC‐L) cells defective in type I IFN secretion indicated that the upregulation of RIG‐I expression in pDCs upon stimulation by endosomal TLR occurs in a type I IFN‐independent manner. Selective phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 could be identified as an early signaling event in this process. Our results show that in contrast to many other cell types, where RIG‐I expression is induced by type I IFN, in pDCs a disparate mechanism is responsible for the upregulation of RIG‐I. Our findings also indicate that along with autophagy, an additional mechanism is operating in pDCs to promote the detection of replicating viruses.

The Two-Component Adjuvant IC31® Boosts Type I Interferon Production of Human Monocyte-Derived Dendritic Cells via Ligation of Endosomal TLRs

The aim of this study was to characterize and identify the mode of action of IC31®, a two-component vaccine adjuvant. We found that IC31® was accumulated in human peripheral blood monocytes, MHC class II positive cells and monocyte-derived DCs (moDCs) but not in plasmacytoid DCs (pDCs). In the presence of IC31® the differentiation of inflammatory CD1a+ moDCs and the secretion of chemokines, TNF-α and IL-6 cytokines was inhibited but the production of IFNβ was increased. Sustained addition of IC31® to differentiating moDCs interfered with IκBα phosphorylation, while the level of phospho-IRF3 increased. We also showed that both IC31® and its KLK component exhibited a booster effect on type I IFN responses induced by the specific ligands of TLR3 or TLR7/8, whereas TLR9 ligand induces type I IFN production only in the presence of IC31® or ODN1. Furthermore, long term incubation of moDCs with IC31® caused significantly higher expression of IRF and IFN genes than a single 24 hr treatment. The adjuvant activity of IC31® on the IFN response was shown to be exerted through TLRs residing in the vesicular compartment of moDCs. Based on these results IC31® was identified as a moDC modulatory adjuvant that sets the balance of the NF-κB and IRF3 mediated signaling pathways to the production of IFNβ. Thus IC31® is emerging as a potent adjuvant to increase immune responses against intracellular pathogens and cancer in future vaccination strategies.

Exposure to inhomogeneous static magnetic field beneficially affects allergic inflammation in a murine model

Previous observations suggest that static magnetic field (SMF)-exposure acts on living organisms partly through reactive oxygen species (ROS) reactions. In this study, we aimed to define the impact of SMF-exposure on ragweed pollen extract (RWPE)-induced allergic inflammation closely associated with oxidative stress. Inhomogeneous SMF was generated with an apparatus validated previously providing a peak-to-peak magnetic induction of the dominant SMF component 389 mT by 39 T m−1 lateral gradient in the in vivo and in vitro experiments, and 192 mT by 19 T m−1 in the human study at the 3 mm target distance. Effects of SMF-exposure were studied in a murine model of allergic inflammation and also in human provoked skin allergy. We found that even a single 30-min exposure of mice to SMF immediately following intranasal RWPE challenge significantly lowered the increase in the total antioxidant capacity of the airways and decreased allergic inflammation. Repeated (on 3 consecutive days) or prolonged (60 min) exposure to SMF after RWPE challenge decreased the severity of allergic responses more efficiently than a single 30-min treatment. SMF-exposure did not alter ROS production by RWPE under cell-free conditions, while diminished RWPE-induced increase in the ROS levels in A549 epithelial cells. Results of the human skin prick tests indicated that SMF-exposure had no significant direct effect on provoked mast cell degranulation. The observed beneficial effects of SMF are likely owing to the mobilization of cellular ROS-eliminating mechanisms rather than direct modulation of ROS production by pollen NAD(P)H oxidases.

Identification of plasmacytoid pre-dendritic cells by one-color flow cytometry for phenotype screening.

Plasmacytoid pre‐dendritic cells (pDCs) are able to prime and polarize naive T‐cells, while also having an important effector function in antiviral immunity through the rapid and robust production of interferon‐α. The main setback of pDCs investigation is the rarity and ex vivo fragility of these cells. Relative simple, reliable, and accurate methods for phenotypic analysis and functional studies of pDCs without isolation would be a great deal of interest. Fresh whole blood samples were analyzed by two‐color and one‐color flow cytometric pDC‐identification assays. The changes in the surface expression of CD62L and HLA‐DQ on pDCs in whole blood samples after 24‐h treatment with imiquimod, a toll‐like receptor 7 agonist, were analyzed. Our data demonstrate that the identification of pDCs in peripheral blood samples can be achieved by using only one fluorescent channel for blood dendritic cell antigen (BDCA)‐4 staining combined with the light scatter parameters, thus leaving the other channels open for further phenotypic and/or functional analysis. Recently, several lines of evidence supported the involvement of pDCs in the development of several human diseases, so our new one‐color identification approach may provide a useful tool for investigation of the pathomechanism of the relevant diseases by using common, 2‐laser benchtop cytometers.

Diet-induced obesity alters dural CGRP release and potentiates TRPA1-mediated trigeminovascular responses

Background Clinical studies suggest a link between obesity and the primary headache disorder migraine. In our study we aimed to reveal the effect of obesity on meningeal nociceptor function in rats receiving a high-fat, high-sucrose diet. Methods Transient receptor potential ankyrin 1 (TRPA1) receptor activation-induced changes in meningeal blood flow, release of calcitonin gene-related peptide (CGRP) from trigeminal afferents and TRPA1 protein expression in the trigeminal ganglia were measured in control and obese rats. Metabolic parameters of the animals were assessed by measuring glucose and insulin homeostasis as well as plasma cytokine concentrations. Results The present experiments revealed an enhanced basal and TRPA1 receptor agonist-induced CGRP release from meningeal afferents of obese insulin-resistant rats and an attenuated CGRP release to potassium chloride. Obesity was also associated with an augmented vasodilatation in meningeal arteries after dural application of the TRPA1 agonist acrolein, a reduction in TRPA1 protein expression in the trigeminal ganglia and elevations in circulating proinflammatory cytokines IL-1β and IL-6 in addition to increased fasting blood glucose and insulin concentrations. Conclusions Our results suggest trigeminal sensitisation as a mechanism for enhanced headache susceptibility in obese individuals after chemical exposure of trigeminal nociceptors.

The antiviral immune response in human conventional dendritic cells is controlled by the mammalian target of rapamycin.

Type I and III IFNs are crucial, soluble components of potent antiviral responses. It has been explored recently that mTOR is involved in the regulation of IFN‐α/β production by pDCs, albeit its role in the induction of IFN responses in cDCs remained unrevealed. In this study, we demonstrate that the PI3K/mTOR pathway is indispensable for eliciting intact type I and III IFN responses in moDCs stimulated with polyI:C. The inhibition of mTOR functionality by rapamycin impairs the pIRF3 and also a few members of the MAPK family, suggesting that mTOR contributes to the activation of multiple signaling pathways in the presence of viral antigens. Furthermore, rapamycin‐treated moDCs show decreased capacity to prime IFN‐γ secretion by naive CD8+ T‐lymphocytes. As in moDCs, mTOR‐mediated regulation is also essential for the production of type I and III IFNs in circulating CD1c+ DCs. To our best knowledge, these results demonstrate for the first time that mTOR has an impact on the functional activities of cDCs via modulating the outcome of IFN secretion.

Immunomodulatory capacity of the serotonin receptor 5-HT2B in a subset of human dendritic cells

Serotonin is a monoamine neurotransmitter that signals through a wide array of receptors (5-HT1–7) many of which are also involved in immune processes. Dendritic cells (DCs) are crucial players in immune defense by bridging innate and adaptive immune responses via their vast repertoire of pattern recognition receptors and antigen-presenting capability. Although serotonin is known to influence immunity at many levels, cell type-specific expression and function of its receptors remains poorly understood. Here we aimed to study 5-HT1–7 expression and function in CD1a− and CD1a+ human monocyte-derived DCs (moDCs). We found that the 5-HT2B receptor-subtype is solely expressed by the inflammatory CD1a+ moDC subset. Specific 5-HT2B activation potently inhibited TLR2, TLR3, and TLR7/8-induced proinflammatory cytokine and chemokine (TNF-α, IL-6, IL-8, IP-10, IL-12) but not type I interferon-β responses. 5-HT2B agonism also interfered with the polarization of CD1a+ moDC-primed CD4+ T cells towards inflammatory Th1 and Th17 effector lymphocytes. Here we report the subset-specific expression and immunomodulatory function of 5-HT2B in human moDCs. Our results expand the biological role of 5-HT2B which may act not only as a neurotransmitter receptor, but also as an important modulator of both innate and adaptive immune responses.

Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.