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Biomedical subjects

Hermann Wagner

Publications and source records attributed to Hermann Wagner.

At least 19 recordsLinked to original sources

Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease.

The scurfy mutant mouse strain suffers from a fatal lymphoproliferative disease leading to early death within 3-4 wk of age. A frame-shift mutation of the forkhead box transcription factor Foxp3 has been identified as the molecular cause of this multiorgan autoimmune disease. Foxp3 is a central control element in the development and function of regulatory T cells (T reg cells), which are necessary for the maintenance of self-tolerance. However, it is unclear whether dysfunction or a lack of T reg cells is etiologically involved in scurfy pathogenesis and its human correlate, the IPEX syndrome. We describe the generation of bacterial artificial chromosome-transgenic mice termed "depletion of regulatory T cell" (DEREG) mice expressing a diphtheria toxin (DT) receptor-enhanced green fluorescent protein fusion protein under the control of the foxp3 gene locus, allowing selective and efficient depletion of Foxp3+ T reg cells by DT injection. Ablation of Foxp3+ T reg cells in newborn DEREG mice led to the development of scurfy-like symptoms with splenomegaly, lymphadenopathy, insulitis, and severe skin inflammation. Thus, these data provide experimental evidence that the absence of Foxp3+ T reg cells is indeed sufficient to induce a scurfy-like phenotype. Furthermore, DEREG mice will allow a more precise definition of the function of Foxp3+ T reg cells in immune reactions in vivo.

Animals↗

Sensitivity to interaural time difference and representation of azimuth in central nucleus of inferior colliculus in the barn owl.

Standard electrophysiology and virtual auditory stimuli were used to investigate the influence of interaural time difference on the azimuthal tuning of neurons in the core and the lateral shell of the central nucleus of the inferior colliculus of the barn owl. The responses of the neurons to virtual azimuthal stimuli depended in a periodic way on azimuth. Fixation of the interaural time difference, while leaving all other spatial cues unchanged, caused a loss of periodicity and a broadening of azimuthal tuning. This effect was studied in more detail in neurons of the core. The azimuthal range tested and the frequency selectivity of the neurons were additional parameters influencing the changes induced by fixating the interaural time difference. The addition of an interaural time difference to the virtual stimuli resulted in a shift of the tuning curves that correlated with the interaural time difference added. In this condition, tuning strength did not change. These results suggest that interaural time difference is an important determinant of azimuthal tuning in all neurons of the core and lateral shell of the central nucleus of the inferior colliculus, and is the only determinant in many of the neurons from the core.

Acoustic Stimulation↗

Yersinia enterocolitica YopP inhibits MAP kinase-mediated antigen uptake in dendritic cells.

Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild-type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)-deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP-C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin-mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA-specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits this pathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity.

Animals↗

The NF-kappaB regulator Bcl-3 and the BH3-only proteins Bim and Puma control the death of activated T cells.

Apoptosis of activated T cells is critical for the termination of immune responses. Here we show that adjuvant-stimulated dendritic cells secrete cytokines that prime activated T cells for survival and analyze the roles of the NF-kappaB regulator Bcl-3 and the proapoptotic Bcl-2 family members Bim and Puma. Bcl-3 overexpression increased survival, and activated bcl-3-/- T cells died abnormally rapidly. Cytokines from adjuvant-stimulated dendritic cells induced Bcl-3, but survival through cytokine priming was Bcl-3-independent. Apoptosis inhibition by Bcl-3 involved blockade of Bim activation, because Bim was overactivated in Bcl-3-deficient cells, and Bcl-3 failed to increase survival of bim-/- T cells. However, adjuvants increased survival also in Bim-deficient T cells. This Bim-independent death pathway is at least in part regulated by Puma, as shown by analysis of puma-/- and noxa-/- T cells. IL-1, IL-7, and IL-15 primed T cells for survival even in the absence of Bim or Puma. Our data define interrelations and a Bim-independent pathway to activated T cell death.

Adjuvants, Immunologic↗

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells.

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFalpha, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFalpha and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.

Animals↗

Toll-like receptor 9-independent aggravation of glomerulonephritis in a novel model of SLE.

The generation of anti-DNA auto-antibodies is characteristic for the human autoimmune condition systemic lupus erythematosus (SLE) and its animal models. However, the contribution of the toll-like receptor (TLR) system of innate immunity receptors and, in particular, TLR9 to this B cell-mediated autoimmune process remains controversial. Here we report that in a novel murine model of SLE, based on hyper-reactive B cell activation mediated by mutant phospholipase Cg2, the genetic deficiency of TLR9 does not protect from spontaneous anti-DNA auto-antibody formation and glomerulonephritis. On the contrary, disease induction is aggravated and additional nucleolar antibody specificity develops in autoimmune TLR9-deficient mice. In vitro studies demonstrate that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may promote autoimmunity, while TLR9 signaling can ameliorate SLE-like immune pathology in vivo.

Animals↗

Influence of the facial ruff on the sound-receiving characteristics of the barn owl's ears.

The barn owl, a nocturnal predator, derives its German name ("Schleiereule", direct English translation "veil owl") from the conspicuous ruff that covers the ear openings and gives the head a face-like appearance. The ruff is a specialization for the perception of sound. The densely-ramified reflector feathers forming the border of the ruff direct sound to the ear-openings. We studied the influence of the ruff on the behaviorally relevant sound-localization parameters interaural time difference (ITD) and interaural level difference (ILD). The directionality of the ear was much greater when the ruff was intact than when the reflector feathers were removed. With ruff intact, the distribution of ILDs was oblique and the maximum ITD occurred around 110 degrees of azimuth. When all head feathers were removed, the steepest ILD gradient was much closer to the horizontal axis and ITD was maximal at 90 degrees . Many effects were frequency specific. Thus, the ruff reflects some properties of the human pinna. However, by shifting the point where ITD becomes maximal beyond 90 degrees , the ruff also introduces a break of the front-back symmetry of ITD.

Acoustic Stimulation↗

On the barn owl's visual pre-attack behavior: I. Structure of head movements and motion patterns.

Barn owls exhibit a rich repertoire of head movements before taking off for prey capture. These movements occur mainly at light levels that allow for the visual detection of prey. To investigate these movements and their functional relevance, we filmed the pre-attack behavior of barn owls. Off-line image analysis enabled reconstruction of all six degrees of freedom of head movements. Three categories of head movements were observed: fixations, head translations and head rotations. The observed rotations contained a translational component. Head rotations did not follow Listing's law, but could be well described by a second-order surface, which indicated that they are in close agreement with Donder's law. Head translations did not contain any significant rotational components. Translations were further segmented into straight-line and curved paths. Translations along an axis perpendicular to the line of sight were similar to peering movements observed in other animals. We suggest that these basic motion elements (fixations, head rotations, translations along a straight line, and translation along a curved trajectory) may be combined to form longer and more complex behavior. We speculate that these head movements mainly underlie estimation of distance during prey capture.

Animals↗

All is not Toll: new pathways in DNA recognition.

Immunological defense depends on the ability of the innate immune system to recognize invading microbes as foreign and thus eliminate them. The Toll-like receptors (TLRs) help detect foreign invaders by sensing various pathogen-associated molecules, including microbial RNA and DNA. At present, it is unclear whether and how the immune system distinguishes between microbial and self nucleic acids, as host-derived RNA and DNA also stimulate TLRs. In addition, recent studies have revealed the existence of TLR-independent pathways that are activated in response to microbial and host nucleic acids.

Animals↗

Differential involvement of TLR2 and TLR4 in host survival during pulmonary infection with Chlamydia pneumoniae.

The relevance of TLR2 and TLR4 for recognizing Chlamydia pneumoniae in vivo during pulmonary infection and to survive the infection was explored. We found that early immune responses triggered by C. pneumoniae partially depended on TLR2, but not on TLR4. The chemokines MIP-2 and MIP-1alpha were not induced, while IL-12p40 levels were higher in TLR2(-/-) mice compared to wild-type mice. Secretion of TNF, keratinocyte-derived chemokine and monocyte chemoattractant protein-1 was attenuated in TLR2(-/-) mice, while IFN-gamma was increased as in wild-type mice. The pulmonary cyto- and chemokine response of TLR2(-/-) x TLR4(d/d) was similar to TLR2(-/-) mice. TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice also attracted fewer polymorphonuclear neutrophils into the lung, while TLR4(d/d) mice recruited them. Attenuated recruitment of polymorphonuclear neutrophils correlated with reduced weight loss in TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice and a lower chlamydial burden 3 days post infection. At 9 days post infection, TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice produced cyto- and chemokines as efficiently as wild-type mice, indicating that the involvement of TLR in inflammation varies over time. All TLR2(-/-) x TLR4(d/d) mice succumbed to the infection, while about 50% of TLR2(-/-) mice died. Taken together, the function of TLR2 and TLR4 is required to survive pulmonary infection with C. pneumoniae.

Adaptor Proteins, Signal Transducing↗

CpG motif-independent activation of TLR9 upon endosomal translocation of "natural" phosphodiester DNA.

Endosomally translocated host (self) DNA activates Toll-like receptor 9 (TLR9), while extracellular self-DNA does not. This inconsistency reflects poor endosomal DNA translocation but also implies that host DNA contains DNA sequences that function as ligands for TLR9. Herein we report that contrary to phosphorothioate (PS)-stabilized oligonucleotides (ODN), "natural" phosphodiester (PD) ODN lacking CpG motifs activate TLR9. CpG motif-independent TLR9 activation of Flt3-L-induced dendritic cells (DC) was dependent on enforced endosomal translocation and triggered upregulation of CD40 and CD69 as well as production of IL-6 and IFN-alpha. Binding studies utilizing surface plasmon resonance technology (Biacore) revealed low TLR9 binding to single-stranded (ss) PD-ODN lacking CpG motifs. At higher concentrations their TLR9 binding activity compared well with TLR9 binding of canonical ss PD CpG-ODN. These results imply that both the chemical modification of the DNA backbone as well as the amount of endosomally translocated DNA represent determining factors that allow CpG motif-independent activation of TLR9 by ss PD-DNA.

Animals↗

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO.

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions.

Adenoviridae↗

Unidirectional development of CD8+ central memory T cells into protective Listeria-specific effector memory T cells.

Three distinct subsets of antigen-experienced CD8(+) T cells have been identified so far: short-living effector T cells (T(EC)) and two long-living subsets, described as central (T(CM)) and effector memory (T(EM)) T cells. The lineage relationships of these subpopulations as well as their involvement in protection have not yet been conclusively determined. We recently described a novel marker combination (CD127 and CD62L) to identify all three major CD8(+) T cell subsets in mice infected with Listeria monocytogenes (L.m.). Extensive lineage relationship analyses on highly purified subpopulations after in vitro and in vivo stimulation demonstrated that T(CM) can develop into T(EM) or T(EC), whereas T(EM) can only progress to T(EC) cells. Short-living T(EC) never regained a T(EM) or T(CM) phenotype. These data strongly suggest a hierarchical and unidirectional order of developmental stages. In vivo priming protocols that preferentially induced one of the different CD8(+) T cell subsets demonstrated that predominance of T(EM) (CD40 stimulation) correlated best with effective protection against L.m., whereas generation of neither T(CM) (by immunization with heat-killed L.m.) nor T(EC) (by systemic co-administration of CpG during primary infection) conferred substantial long-term protective immunity. These findings have important implications for the design of more effective T cell-based vaccines.

Animals↗

Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production.

BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches.

Adenoviridae↗

Endogenous TLR ligands and autoimmunity.

Based on an evolutionary conserved repertoire Toll-like-receptors (TLRs) donate specificity to innate immune cells. Therefore, TLRs are considered as paradigmatic for "self" versus "non-self" discrimination. This view, however, needs to be modified since TLR's also appear to recognise "endogeneous", that is host-derived ligands, examples being host-derived DNA and -RNA. Here I discuss physiological and pathophysiological consequences of endogeneous ligand-recognition by TLRs. I conclude that endogeneous ligand recognition by TLRs drives sterile inflammation sustained by innate immune cells in certain autoimmune disorders.

Animals↗

CD25+/Foxp3+ T cells regulate gastric inflammation and Helicobacter pylori colonization in vivo.

BACKGROUND & AIMS: Helicobacter pylori infects more than half of the world's population. In contrast to most other pathogens, the microbe persists for the virtual life of its host. It is unclear why the immune system is unable to eliminate the infection, but recent studies suggested that CD4+/CD25+/Foxp3+ regulatory T cells may be involved in this process. METHODS: By using a mouse model of infection and gastric biopsies from 108 patients, we performed a detailed descriptive and functional characterization of the Helicobacter-induced CD25+/Foxp3+ T-cell response. RESULTS: In C57BL/6 mice, H pylori induced a marked gastric Foxp3+ T-cell response, which increased over several months together with the severity of inflammation, until a stable homeostatic situation became established. Accordingly, in Helicobacter-infected patients, but not in uninfected individuals, large numbers of gastric Foxp3+ T cells were detected immunohistochemically. To define the functional in vivo relevance of this response, CD25+ cells were depleted systemically in mice by using an anti-CD25 monoclonal antibody (PC61). Already 4 weeks after infection, PC61-treated mice, but not untreated animals, developed a severe gastritis with heightened cytokine expression and increased numbers of mucosal T cells, B cells, and macrophages. This was accompanied by increased titers of H pylori-specific IgG1 and IgG2c antibodies in the sera of PC61-treated mice. This increased gastric inflammatory response in CD25-depleted mice was associated with reduced bacterial loads. CONCLUSIONS: CD25+/Foxp3+ T cells actively participate in the immune response to H pylori. In vivo depletion of these cells in infected mice leads to increased gastric inflammation and reduced bacterial colonization.

Animals↗

Role of immunostimulatory DNA and TLR9 in gene therapy.

One of the key issues of DNA-based therapies is the immunostimulatory effect caused by DNA, which it has a significant impact on the consequences of these therapies. This is a result of the recognition by Toll-like receptor-9 (TLR9); a pattern recognition receptor for unmethylated CpG motifs within DNA sequences. However, recent studies show that TLR9 can recognize non-CpG motifs and induce cellular activation when DNA is efficiently delivered to TLR9, for example, by cationic lipids. Moreover, recent research shows that TLR9-independent immunoactivation could take place. Here, we have attempted to present an overview of immunoactivation by DNA to optimize DNA-based therapies.

Adenoviridae↗

U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7.

Plasmacytoid dendritic cells (PDCs), which produce IFN-alpha in response to autoimmune complexes containing nuclear antigens, are thought to be critically involved in the pathogenesis of systemic lupus erythematosus (SLE). One of the immunostimulatory components of SLE immune complexes (SLE-ICs) is self DNA, which is recognized through Tlr9 in PDCs and B cells. Small nuclear ribonucleoproteins (snRNPs) are another major component of SLE-ICs in 30% to 40% of patients. In this study, we show that murine PDCs are activated by purified U1snRNP/anti-Sm ICs to produce IFN-alpha and proinflammatory cytokines and to up-regulate costimulatory molecules. The induction of IFN-alpha and IL-6 by U1snRNPs in murine bone marrow-derived PDCs required the presence of intact U1RNA and was largely dependent on Tlr7 but independent of Tlr3. Intracellularly delivered isolated U1snRNA and oligoribonucleotides derived from the stem loop regions and the Sm-binding site of U1snRNA efficiently induced IFN-alpha and IL-6 in Flt3L-cultured DCs in a Tlr7-dependent manner. The U1snRNA component of U1snRNP immune complexes, found in patients with SLE, acts as an endogenous "self" ligand for Tlr7 and triggers IFN-alpha and IL-6 production in PDCs.

Animals↗