Radiation-Detoxified Form of Endotoxin Effectively Activates Th1 Responses and Attenuates Ragweed-Induced Th2-Type Airway Inflammation in Mice.

Urbanization with reduced microbial exposure is associated with an increased burden of asthma and atopic symptoms. Conversely, environmental exposure to endotoxins in childhood can protect against the development of allergies. Our study aimed to investigate whether the renaturation of the indoor environment with aerosolized radiation-detoxified lipopolysaccharide (RD-LPS) has a preventative effect against the development of ragweed-induced Th2-type airway inflammation. To explore this, cages of six-week-old BALB/c mice were treated daily with aerosolized native LPS (N-LPS) or RD-LPS. After a 10-week treatment period, mice were sensitized and challenged with ragweed pollen extract, and inflammatory cell infiltration into the airways was observed. As dendritic cells (DCs) play a crucial role in the polarization of T-cell responses, in our in vitro experiments, the effects of N-LPS and RD-LPS were compared on human monocyte-derived DCs (moDCs). Mice in RD-LPS-rich milieu developed significantly less allergic airway inflammation than mice in N-LPS-rich or common environments. The results of our in vitro experiments demonstrate that RD-LPS-exposed moDCs have a higher Th1-polarizing capacity than moDCs exposed to N-LPS. Consequently, we suppose that the aerosolized, non-toxic RD-LPS applied in early life for the renaturation of urban indoors may be suitable for the prevention of Th2-mediated allergies in childhood.

Type I Interferon Production of Plasmacytoid Dendritic Cells under Control.

One of the most powerful and multifaceted cytokines produced by immune cells are type I interferons (IFNs), the basal secretion of which contributes to the maintenance of immune homeostasis, while their activation-induced production is essential to effective immune responses. Although, each cell is capable of producing type I IFNs, plasmacytoid dendritic cells (pDCs) possess a unique ability to rapidly produce large amounts of them. Importantly, type I IFNs have a prominent role in the pathomechanism of various pDC-associated diseases. Deficiency in type I IFN production increases the risk of more severe viral infections and the development of certain allergic reactions, and supports tumor resistance; nevertheless, its overproduction promotes autoimmune reactions. Therefore, the tight regulation of type I IFN responses of pDCs is essential to maintain an adequate level of immune response without causing adverse effects. Here, our goal was to summarize those endogenous factors that can influence the type I IFN responses of pDCs, and thus might serve as possible therapeutic targets in pDC-associated diseases. Furthermore, we briefly discuss the current therapeutic approaches targeting the pDC-type I IFN axis in viral infections, cancer, autoimmunity, and allergy, together with their limitations defined by the Janus-faced nature of pDC-derived type I IFNs.

Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR.

To detect replicating viruses, dendritic cells (DCs) utilize cytoplasmic retinoic acid inducible gene-(RIG) I-like receptors (RLRs), which play an essential role in the subsequent activation of antiviral immune responses. In this study, we aimed to explore the role of the mammalian target of rapamycin (mTOR) in the regulation of RLR-triggered effector functions of human monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs). Our results show that RLR stimulation increased the phosphorylation of the mTOR complex (mTORC) 1 and mTORC2 downstream targets p70S6 kinase and Akt, respectively, and this process was prevented by the mTORC1 inhibitor rapamycin as well as the dual mTORC1/C2 kinase inhibitor AZD8055 in both DC subtypes. Furthermore, inhibition of mTOR in moDCs impaired the RLR stimulation-triggered glycolytic switch, which was reflected by the inhibition of lactate production and downregulation of key glycolytic genes. Blockade of mTOR diminished the ability of RLR-stimulated moDCs and pDCs to secret type I interferons (IFNs) and pro-inflammatory cytokines, while it did not affect the phenotype of DCs. We also found that mTOR blockade decreased the phosphorylation of Tank-binding kinase 1 (TBK1), which mediates RLR-driven cytokine production. In addition, rapamycin abrogated the ability of both DC subtypes to promote the proliferation and differentiation of IFN-y and Granzyme B producing CD8 + T cells. Interestingly, AZD8055 was much weaker in its ability to decrease the T cell proliferation capacity of DCs and was unable to inhibit the DC-triggered production of IFN-y and Granyzme B by CD8 + T cells. Here we demonstrated for the first time that mTOR positively regulates the RLR-mediated antiviral activity of human DCs. Further, we show that only selective inhibition of mTORC1 but not dual mTORC1/C2 blockade suppresses effectively the T cell stimulatory capacity of DCs that should be considered in the development of new generation mTOR inhibitors and in the improvement of DC-based vaccines.

Signaling Lymphocyte Activation Molecule Family 5 Enhances Autophagy and Fine-Tunes Cytokine Response in Monocyte-Derived Dendritic Cells via Stabilization of Interferon Regulatory Factor 8.

Signaling lymphocyte activation molecule family (SLAMF) receptors are essential regulators of innate and adaptive immune responses. The function of SLAMF5/CD84, a family member with almost ubiquitous expression within the hematopoietic lineage is poorly defined. In this article, we provide evidence that in human monocyte-derived dendritic cells (moDCs) SLAMF5 increases autophagy, a degradative pathway, which is highly active in dendritic cells (DCs) and plays a critical role in orchestration of the immune response. While investigating the underlying mechanism, we found that SLAMF5 inhibited proteolytic degradation of interferon regulatory factor 8 (IRF8) a master regulator of the autophagy process by a mechanism dependent on the E3-ubiquitin ligase tripartite motif-containing protein 21 (TRIM21). Furthermore, we demonstrate that SLAMF5 influences the ratio of CD1a+ cells in differentiating DCs and partakes in the regulation of IL-1ß, IL-23, and IL-12 production in LPS/IFNγ-activated moDCs in a manner that is consistent with its effect on IRF8 stability. In summary, our experiments identified SLAMF5 as a novel cell surface receptor modulator of autophagy and revealed an unexpected link between the SLAMF and IRF8 signaling pathways, both implicated in multiple human pathologies.

Human Plasmacytoid and Monocyte-Derived Dendritic Cells Display Distinct Metabolic Profile Upon RIG-I Activation.

Recent advances reveal that metabolic reprogramming is required for adequate antiviral responses of dendritic cells (DCs) that possess the capacity to initiate innate and adaptive immune responses. Several reports indicate that Toll-like receptor (TLR) stimulation of DCs is accompanied by a rapid induction of glycolysis; however, the metabolic requirements of retinoic-acid inducible gene I (RIG-I)-like receptor (RLR) activation have not defined either in conventional DCs (cDCs) or in plasmacytoid DCs (pDCs) that are the major producers of type I interferons (IFN) upon viral infections. To sense viruses and trigger an early type I IFN response, pDCs rely on endosomal TLRs, whereas cDCs employ cytosolic RIG-I, which is constitutively present in their cytoplasm. We previously found that RIG-I is upregulated in pDCs upon endosomal TLR activation and contributes to the late phase of type I IFN responses. Here we report that TLR9-driven activation of human pDCs leads to a metabolic transition to glycolysis supporting the production of type I IFNs, whereas RIG-I-mediated antiviral responses of pDCs do not require glycolysis and rather rely on oxidative phosphorylation (OXPHOS) activity. In particular, TLR9-activated pDCs show increased extracellular acidification rate (ECAR), lactate production, and upregulation of key glycolytic genes indicating an elevation in glycolytic flux. Furthermore, administration of 2-deoxy-D-glucose (2-DG), an inhibitor of glycolysis, significantly impairs the TLR9-induced secretion of type I IFNs by human pDCs. In contrast, RIG-I stimulation of pDCs does not result in any alterations of ECAR, and type I IFN production is not inhibited but rather promoted by 2-DG treatment. Moreover, pDCs activated via TLR9 but not RIG-I in the presence of 2-DG are impaired in their capacity to prime allogeneic naïve CD8+ T cell proliferation. Interestingly, human monocyte-derived DCs (moDC) triggered via RIG-I show a commitment to glycolysis to promote type I IFN production and T cell priming in contrast to pDCs. Our findings reveal for the first time, that pDCs display a unique metabolic profile; TLR9-driven but not RIG-I-mediated activation of pDCs requires glycolytic reprogramming. Nevertheless, the metabolic signature of RIG-I-stimulated moDCs is characterized by glycolysis suggesting that RIG-I-induced metabolic alterations are rather cell type-specific and not receptor-specific.

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.

Commercial strain-derived clinical Saccharomyces cerevisiae can evolve new phenotypes without higher pathogenicity.

SCOPE: Saccharomyces cerevisiae is one of the most important microbes in food industry, but there is growing evidence on its potential pathogenicity as well. Its status as a member of human mycobiome is still not fully understood. METHODS AND RESULTS: In this study, we characterize clinical S. cerevisiae isolates from Hungarian hospitals along with commercial baking and probiotic strains, and determine their phenotypic parameters, virulence factors, interactions with human macrophages, and pathogenicity. Four of the clinical isolates could be traced back to commercial strains based on genetic fingerprinting. Our observations indicate that the commercial-derived clinical isolates have evolved new phenotypes and show similar, or in two cases, significantly decreased pathogenicity. Furthermore, immunological experiments revealed that the variability in human primary macrophage activation after coincubation with yeasts is largely donor and not isolate dependent. CONCLUSION: Isolates in this study offer an interesting insight into the potential microevolution of probiotic and food strains in human hosts. These commensal yeasts display various changes in their phenotypes, indicating that the colonization of the host does not necessarily impose a selective pressure toward higher virulence/pathogenicity.

Diet-Induced Obesity Enhances TRPV1-Mediated Neurovascular Reactions in the Dura Mater.

OBJECTIVE: Exploring the pathophysiological changes in transient receptor potential vanilloid 1 (TRPV1) receptor of the trigeminovascular system in high-fat, high-sucrose (HFHS) diet-induced obesity of experimental animals. BACKGROUND: Clinical and experimental observations suggest a link between obesity and migraine. Accumulating evidence indicates that metabolic and immunological alterations associated with obesity may potentially modulate trigeminovascular functions. A possible target for obesity-induced pathophysiological changes is the TRPV1/capsaicin receptor which is implicated in the pathomechanism of headaches in a complex way. METHODS: Male Sprague-Dawley rats were fed a regular (n = 25) or HFHS diet (n = 26) for 20 weeks. At the end of the dietary period, body weight of the animals was normally distributed in both groups and it was significantly higher in animals on HFHS diet. Therefore, experimental groups were regarded as control and HFHS diet-induced obese groups. Capsaicin-induced changes in meningeal blood flow and release of calcitonin gene-related peptide (CGRP) from dural trigeminal afferents were measured in control and obese rats. The distribution of TRPV1- and CGRP-immunoreactive meningeal sensory nerves was also compared in whole mount preparations of the dura mater. Metabolic parameters of the animals were assessed by examining glucose and insulin homeostasis as well as plasma cytokine concentrations. RESULTS: HFHS diet was accompanied by reduced food consumption and greater fluid and energy intakes in addition to increased body weight of the animals. HFHS diet increased fasting blood glucose and insulin concentrations as well as levels of circulating proinflammatory cytokines interleukin-1ß and interleukin-6. In obese animals, dural application of the archetypal TRPV1 agonist capsaicin resulted in significantly augmented vasodilatory and vasoconstrictor responses as compared to controls. Diet-induced obesity was also associated with enhanced basal and capsaicin-induced CGRP release from meningeal afferents ex vivo. Except for minor morphological changes, the distribution of dural TRPV1- and CGRP-immunoreactive afferents was similar in control and obese animals. CONCLUSIONS: Our results suggest that obesity induced by long-term HFHS diet results in sensitization of the trigeminovascular system. Changes in TRPV1-mediated vascular reactions and CGRP release are pathophysiological alterations that may be of relevance to the enhanced headache susceptibility of obese individuals.

RIG-I inhibits the MAPK-dependent proliferation of BRAF mutant melanoma cells via MKP-1.

BACKGROUND: BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. METHODS: Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. RESULTS: Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNß pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/ß, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. CONCLUSION: RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNß production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.

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/ß 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 IFNß 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 IFNß. Thus IC31® is emerging as a potent adjuvant to increase immune responses against intracellular pathogens and cancer in future vaccination strategies.

Ragweed subpollen particles of respirable size activate human dendritic cells.

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3(+) pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.

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-alpha 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.