Signaltransduktion von IgG-Antiphospholipid-Antikörpern in plasmazytoiden Dendriten und Monomac1 Zellen

Das Antiphospholipid-Syndrom (APS) ist eine Autoimmunerkrankung die sich durch venöse und arterielle Thrombosen und/oder Spontanaborte bei gleichzeitigem Nachweis von persistierenden, erhöhten Antiphospholipid-Antikörper (aPL)-Titern charakterisieren lässt. Die zugrunde liegenden Mechanismen, über die aPL Pathogenität vermitteln, sind bislang wenig verstanden. Im Rahmen dieser Arbeit konnte gezeigt werden, dass drei humane monoklonale IgG aPL sowie IgG Fraktionen von APS Patienten eine Überexpression von TLR7 und TLR8 in plasmazytoiden dendritischen Zellen bzw. monozytären Zellen induzieren. Gleichzeitig erfolgt die Induktion der TLR7/8 Translokation vom endoplasmatischen Retikulum (ER) ins Endosom. Diese Effekte werden durch die Internalisierung der aPL und die nachfolgende Aktivierung einer NADPH Oxidase sowie durch endosomale Superoxid Produktion vermittelt. Als Folge dessen werden die Zellen extrem für TLR7/8 Liganden sensibilisiert. Diese Beobachtungen beschreiben einen neuen Signalmechanismus der innaten Immunität, der seinen Ursprung im Endosom nimmt. Da die Überexpression von TLR7 auch in pDCs von APS Patienten detektiert werden konnte, bieten unsere Ergebnisse eine Erklärung für die proinflammatorischen und prokoagulanten Effekte von aPL. rnWeiterhin führte die kombinierte Stimulation mit aPL und TLR7 Liganden in pDCs zu einem signifikant verstärkten Potential zur CD4+ Th2 Zell Aktivierung bzw. zur Regulation der B-Zell Differenzierung und Immunglobulin Produktion. Die Anwesenheit der pDCs erhöhte dabei synergistisch die CD40/86 Expression, die Proliferation sowie die Plasmazell-Differenzierung von isolierten peripheren B-Zellen, die mit aPL und TLR Liganden stimuliert wurden. Dieser Stimulationsansatz war außerdem ausreichend um naive B-Zellen zur IgM/IgG Produktion anzuregen und die Synthese neuer IgG aPL durch Gedächtnis-B-Zellen einzuleiten. Die Beteiligung der pDCs an diesem Prozess erfolgte durch Zytokin Sekretion sowie direktem Zell-Zell-Kontakt. Die Anwesenheit von Th2-Helferzellen war dabei nicht obligatorisch, konnte jedoch die B-Zell Aktivierung zusätzlich fördern. Eine Hochregulierung von TLR7 oder TLR9 innerhalb der B-Zell Population war nicht involviert. rnrnDiese Ergebnisse zeigen erstmalig die Relevanz einer pDC Aktivierung im Hinblick auf die Aufrechterhaltung der pathogenen Aktivität im Rahmen des APS. Da eine Dysregulierung von TLR7 bereits als ursächlich für die Ausbildung einer systemischen Autoimmunität erachtet wird, sollten unsere Ergebnisse für das generelle Verständnis von Autoimmunität von großer Relevanz sein.rn

Vergleichende Untersuchungen zur funktionellen Wechselwirkung von T-Helferzellen, regulatorischen T-Zellen und dendritischen Zellen

Dendritische Zellen (DC) verbinden angeborene und adaptive Immunität. Abhängig vom Aktivierungsgrad vermitteln sie Toleranz oder Immunität. Hier wurden konventionelle DC (kDC) und plasmazytoide DC (pDC) hinsichtlich ihrer T-Zellstimulation in An- und Abwesenheit regulatorischer T Zellen (Treg) verglichen. Als starke T-Zellstimulatoren induzieren aktivierte kDC Treg-Proliferation. Proliferierende Treg sind nicht suppressiv, weshalb T-Effektorzellen nach kDC-Stimulation in Treg-Anwesenheit proliferieren. Demgegenüber vermitteln aktivierte pDC nur schwache T-Zellstimulation, die aber ausreicht T-Effektorzellproliferation auszulösen. Aktivierte pDC brechen aber nicht die Anergie von Treg und induzieren daher nicht ihre Proliferation. Trotz der geringen T-Zellstimulation sind pDC in der Lage Proliferation und Expansion von T-Effektorzellen in Treg-Gegenwart zu bewirken. Dies konnte auf ein insuffizientes pDC-vermitteltes T-Zellrezeptorsignal für Treg zurückgeführt werden, das keine funktionelle pDC-vermittelte Treg-Aktivierung bewirkt.rnUnter homöostatischen Bedingung vermitteln DC Toleranz, so induzieren unreife kDC Treg in der Peripherie. Hierbei ist eine ICOS-vermittelte Kostimulation essentiell für die Induktion des IL-10-Rezeptors auf T-Zellen. Ein gleichzeitiges starkes CD28-Signal überkommt den ICOS-vermittelten Effekt und wirkt der IL-10R-Expression entgegen. Daher führt nur die Stimulation mit unreifen kDC zu IL-10-sensitiven T-Zellen in denen dann ein anergischer Phänotyp etabliert wird, der Teil des Differenzierungsprozesses zur induzierten Treg ist. Die ICOS/ICOS-L-Interaktion ist somit zentral für die Aufrechterhaltung peripherer Toleranz.rn

Biomedical nanoparticles modulate specific CD4+ T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4+ T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4+ T cells, and induce cytokines. The decreased antigen processing and CD4+ T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Slc15a4, a gene required for pDC sensing of TLR ligands, is required to control persistent viral infection

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.

Cathepsin S dominates autoantigen processing in human thymic dendritic cells

The interaction of developing thymocytes with peptide-MHC complexes on thymic antigen presenting cells (APC) is crucial for T cell development, both for positive selection of "useful" thymocytes as well as negative selection of autoreactive thymocytes to prevent autoimmunity. The peptides presented on MHC II molecules are generated by lysosomal proteases such as the cathepsins. At the same time, lysosomal proteases will also destroy other potential T cell epitopes from self-antigens. This will lead to a lack of presentation on negatively selecting thymic antigen presenting cells and consequently, escape of autoreactive T cells recognizing these epitopes. In order to understand the processes that govern generation or destruction of self-epitopes in thymic APC, we studied the antigen processing machinery and epitope processing in the human thymus. We find that each type of thymic APC expresses a different signature of lysosomal proteases, providing indirect evidence that positive and negative selection of CD4(+) T cells might occur on different sets of peptides, in analogy to what has been proposed for CD8(+) T cells. We also find that myeloid dendritic cells (DC) are more efficient in processing autoantigen than plasmacytoid DC. In addition, we observed that cathepsin S plays a central role in processing of the autoantigens myelin basic protein and proinsulin in thymic dendritic cells. Cathepsin S destroyed a number of known T cell epitopes, which would be expected to result in lack of presentation and consequently, escape of autoreactive T cells. Cathepsin S therefore appears to be an important factor that influences selection of autoreactive T cells.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Airway epithelial IL-15 transforms monocytes into dendritic cells

IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.

Cathepsin G is differentially expressed in primary human antigen-presenting cells

Cathepsins are required for the processing of antigens in order to make them suitable for loading on major histocompatibility complex (MHC) class II molecules, for subsequent presentation to CD4(+) T cells. It was shown that antigen processing in monocyte-derived dendritic cells (DC), a commonly used DC model, is different from that of primary human DC. Here, we report that the two subsets of human myeloid DC (mDC) and plasmacytoid DC (pDC) differ in their cathepsin distribution. The serine protease cathepsin G (CatG) was detected in mDC1, mDC2, pDC, cortical thymic epithelial cells (cTEC) and high levels of CatG were determined in pDC. To address the role of CatG in the processing and presentation of a Multiple Sclerosis-associated autoantigen myelin basic protein (MBP), we used a non-CatG expressing fibroblast cell line and fibroblasts, which were preloaded with purified CatG. We find that preloading fibroblasts with CatG results in a decrease of MBP84-98-specific T cell proliferation, when compared to control cells. Our data suggest a different processing signature in primary human antigen-presenting cells and CatG may be of functional importance.

Immune recognition of self nucleic acids driven by endogenous antimicrobial peptides: role in autoimmunity

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from enzymatic degradation and transport it into the endosomal compartments of both plasmacytoid and myeloid dendritic cells. In the plasmacytoid DCs, the self-RNA-LL37 complexes activate TLR7 and like the self-DNA-LL37 complexes, trigger the production of IFN-α in the absence of induction of maturation or production of IL-6 and TNF-α. In contrast to the self-DNA-LL37 complexes, the self-RNA-LL37 complexes are also internalized into the endosomal compartments of myeloid dendritic cells and trigger activation through TLR8, leading to the production of TNF-α and IL-6, and the maturation of the myeloid DCs. Furthermore, we found that these self nucleic acid-LL37 complexes can be found in vivo in the skin lesions of the cutaneous autoimmune disease psoriasis, where they are associated with mature mDCs in situ. On the other hand, in the systemic autoimmune disease systemic lupus erythematosus, self-DNA-LL37 complexes were found to be a constituent of the circulating immune complexes isolated from patient sera. This interaction between the endogenous peptide with the self nucleic acid molecules present in the immune complexes was found to be electrostatic and it confers resistance to enzymatic degradation of the nucleic acid molecules in the immune complexes. Moreover, autoantibodies to these endogenous peptides were found to trigger neutrophil activation and release of neutrophil extracellular traps composed of DNA, which are potential sources of the self nucleic acid-LL37 complexes present in SLE immune complexes. Our results demonstrate that the cationic antimicrobial peptide LL37 drives the innate immune recognition of self nucleic acid molecules through toll-like receptors in human dendritic cells, thus elucidating a pathway for innate sensing of host cell death. This pathway of autoreactivity was found to be pathologically relevant in human autoimmune diseases psoriasis and SLE, and thus this study provides new insights into the mechanisms autoimmune diseases.

The immunology of the porcine skin and its value as a model for human skin.

The porcine skin has striking similarities to the human skin in terms of general structure, thickness, hair follicle content, pigmentation, collagen and lipid composition. This has been the basis for numerous studies using the pig as a model for wound healing, transdermal delivery, dermal toxicology, radiation and UVB effects. Considering that the skin also represents an immune organ of utmost importance for health, immune cells present in the skin of the pig will be reviewed. The focus of this review is on dendritic cells, which play a central role in the skin immune system as they serve as sentinels in the skin, which offers a large surface area exposed to the environment. Based on a literature review and original data we propose a classification of porcine dendritic cell subsets in the skin corresponding to the subsets described in the human skin. The equivalent of the human CD141(+) DC subset is CD1a(-)CD4(-)CD172a(-)CADM1(high), that of the CD1c(+) subset is CD1a(+)CD4(-)CD172a(+)CADM1(+/low), and porcine plasmacytoid dendritic cells are CD1a(-)CD4(+)CD172a(+)CADM1(-). CD209 and CD14 could represent markers of inflammatory monocyte-derived cells, either dendritic cells or macrophages. Future studies for example using transriptomic analysis of sorted populations are required to confirm the identity of these cells.

Is The Allergen Really Needed in Allergy Immunotherapy?

Immunotherapy for type I allergies is well established and is regarded to be the most efficient treatment option besides allergen avoidance. As of today, different forms of allergen preparations are used in this regard, as well as different routes of application. Virus-like particles (VLPs) represent a potent vaccine platform with proven immunogenicity and clinical efficacy. The addition of toll-like receptor ligands and/or depot-forming adjuvants further enhances activation of innate as well as adaptive immune responses. CpG motifs represent intensively investigated and potent direct stimulators of plasmacytoid dendritic cells and B cells, while T cell responses are enhanced indirectly through increased antigen presentation and cytokine release. This article will focus on the function of VLPs loaded with DNA rich in nonmethylated CG motifs (CpGs) and the clinical experience gained in the treatment of allergic rhinitis, demonstrating clinical efficacy also if administered without allergens. Several published studies have demonstrated a beneficial impact on allergic symptoms by treatment with CpG-loaded VLPs. Subcutaneous injection of VLPs loaded with CpGs was tested with or without the adjuvant alum in the presence or absence of an allergen. The results encourage further investigation of VLPs and CpG motifs in immunotherapy, either as a stand-alone product or as adjuvants for allergen-specific immunotherapy.

Comparative dendritic cell biology of veterinary mammals.

Dendritic cells (DC) have a main function in innate immunity in that they sense infections and environmental antigens at the skin and mucosal surfaces and thereby critically influence decisions about immune activation or tolerance. As professional antigen-presenting cells, they are essential for induction of adaptive immune responses. Consequently, knowledge on this cell type is required to understand the immune systems of veterinary mammals, including cattle, sheep, pigs, dogs, cats, and horses. Recent ontogenic studies define bona fide DC as an independent lineage of hematopoietic cells originating from a common precursor. Distinct transcription factors control the development into the two subsets of classical DC and plasmacytoid DC. These DC subsets express a distinguishable transcriptome, which differs from that of monocyte-derived DC. Using a comparative approach based on phenotype and function, this review attempts to classify DC of veterinary mammals and to describe important knowledge gaps.

Immune Responses Against Classical Swine Fever Virus: Between Ignorance and Lunacy.

Classical swine fever virus infection of pigs causes disease courses from life-threatening to asymptomatic, depending on the virulence of the virus strain and the immunocompetence of the host. The virus targets immune cells, which are central in orchestrating innate and adaptive immune responses such as macrophages and conventional and plasmacytoid dendritic cells. Here, we review current knowledge and concepts aiming to explain the immunopathogenesis of the disease at both the host and the cellular level. We propose that the interferon type I system and in particular the interaction of the virus with plasmacytoid dendritic cells and macrophages is crucial to understand elements governing the induction of protective rather than pathogenic immune responses. The review also concludes that despite the knowledge available many aspects of classical swine fever immunopathogenesis are still puzzling.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity.

Immune restoration of patients with chronic myelogenous leukemia in complete cytogenetic remission

Imatinib mesylate (IM) and Interferon-alfa (IFN-α) are currently the two most efficacious therapies for patients with chronic myelogenous leukemia (CML). IFN-α induces durable complete cytogentic remission (CCR) in about 25% of CML patients whereas IM, a tyrosine kinase inhibitor, induces CCR in 50% of patients who are resistant to IFN-α and in 75% of patients in early chronic phase of CML. However, the detection of minimal residual disease without clinical relapse suggests that host immune surveillance plays a very important role in controlling the progression of disease. ^ T lymphocytes and dendritic cells (DC) are the two most crucial players in the immune system. In my study, we focused on the effects of treatment with either IM or IFN-α on the functions of both DC and T cells, as exemplified by the ability of DC to present antigen to T cells and activated T cells to synthesize cytokines. Our studies show that cytokine production by T cells activated through the T-cell receptor (TCR) was significantly lower in CML patients treated with IM, but not with IFN-α, when compared with activated T cells of control subjects. Suppression of T cell function by IM albeit transient and reversible, was through the downregulation of the phosphorylation of Zap-70, Lck, and LAT. ^ Our data also show that the myeloid DC (DC1) and the plasmacytoid DC (DC2) are lower in chronic phase CML. Whereas neither therapy restored the level of DC2 to normal levels, the number of DC1 was normalized by either therapy. However, only IFN-α, and not IM, restored DC2 function to normal, as exemplified by the production of IFN-α in response to exposure to live influenza virus. Moreover, in vitro differentiation and maturation of DC1 from monocyte precursors in patients receiving either therapy was not normal and was reflected in their ability to present antigen to autologous T cells. ^ In summary, we report that there are differences in immune responses of CML patients treated with IM or IFN-α that may be the result of long-term effects on the host immune system by the individual therapy. ^