Anna-Maria Dittrich

NEMO Mutations in 2 Unrelated Boys With Severe Infections and Conical Teeth

X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency is a developmental and immunologic disorder caused by mutations in nuclear factor-κB essential modulator (NEMO), which is essential for nuclear factor-κB activation. Early in life, affected boys present a typical appearance, with hypotrichosis or atrichosis, hypohidrosis or anhidrosis, and hypodontia or anodontia with conical incisors. They are also susceptible to various microorganisms, mostly pyogenic bacteria and mycobacteria. Here we report 2 unrelated boys, aged 6 and 11 years, who have novel mutations in NEMO and present conical incisors and hypodontia as their sole and long-unrecognized developmental anomaly. One child had isolated recurrent pneumococcal disease, whereas the other had multiple infections. Our observations indicate that conical incisors should prompt the search for NEMO mutations in boys with unusual infectious diseases.

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

RATIONALE Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood. OBJECTIVES To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages. METHODS Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays. MEASUREMENTS AND MAIN RESULTS Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. CONCLUSIONS These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Glutathione peroxidase-2 protects from allergen-induced airway inflammation in mice

The aim of the present study was to identify and validate the biological significance of new genes/proteins involved in the development of allergic airway disease in a murine asthma model. Gene microarrays were used to identify genes with at least a two-fold increase in gene expression in lungs of two separate mouse strains with high and low allergic susceptibility. Validation of mRNA data was obtained by western blotting and immunohistochemistry, followed by functional analysis of one of the identified genes in mice with targeted disruption of specific gene expression. Expression of two antioxidant enzymes, glutathione peroxidase-2 (GPX2) and glutathione S-transferase omega (GSTO) 1-1 was increased in both mouse strains after induction of allergic airway disease and localised in lung epithelial cells. Mice with targeted disruption of the Gpx-2 gene showed significantly enhanced airway inflammation compared to sensitised and challenged wild-type mice. Our data indicate that genes encoding the antioxidants GPX2 and GSTO 1-1 are common inflammatory genes expressed upon induction of allergic airway inflammation, and independently of allergic susceptibility. Furthermore, we provide evidence to illustrate the importance of a single antioxidant enzyme, GPX2, in protection from allergen-induced disease.

A Toll-Like Receptor 2/6 Agonist Reduces Allergic Airway Inflammation in Chronic Respiratory Sensitisation to Timothy Grass Pollen Antigens

Background: The hygiene hypothesis negatively correlates the microbial burden of the environment with the prevalence of T helper type 2 (Th2)-related disorders, e.g. allergy and asthma. This is explained by Th1 triggering through pathogen-associated molecular patterns via Toll-like receptors (TLRs). In this study, the biological effects of a TLR2/6 agonist as a potential treatment of allergic inflammation are explored. Methods: In a model of chronic allergic airway inflammation induced by intranasal administration of Timothy grass pollen allergen extract, early TLR agonism and/or interferon (IFN)-γ administration was compared to the therapeutic and immune-modulating effects of dexamethasone with regard to the cellular inflammation and cytokine profiles. Results: Eosinophilic inflammation was clearly reduced by TLR2/6 agonism. This effect was also seen without simultaneous administration of IFN-γ. However, lymphocyte counts were not affected among the different treatment groups. More precise determination of the lymphocyte-mediated immune reaction showed that TLR2/6 agonism induced neither CD4+foxp3+ regulatory T cells in draining lymph nodes nor a pronounced Th1 immune response. In contrast, dexamethasone reduced both sensitisation as well as allergic inflammation and, in addition, CD11c+ antigen-presenting cells in lymph nodes. Our data clearly point to the potential to rebalance Th2-skewed allergic immune responses by therapeutic TLR2/6 agonist administration. Conclusion: The use of the TLR2/6 agonist is a promising therapeutic approach in diseases with an imbalance in T cell responses, such as allergy and asthma.

The role of lipocalins in airway disease.

The pathogenesis of allergic airway inflammation and disease is complex and still not fully understood. Many cells, factors and mediators are involved in the different aspects of induction, maintenance and persistence of airway inflammation. The heterogeneity and redundancy of this system is one of the main reasons why novel therapeutic targets focusing on the pathogenesis of asthma only hesitantly reach the market and clinical application. Thus, it seems mandatory that we proceed in our efforts to better understand this micro cosmos to succeed in the development of safe and effective drugs for the treatment of more severe and refractory forms of asthma and chronic obstructive pulmonary disease. One of the more recently discovered mediators in the context of airway inflammation are the lipocalins (Lcns). They are a family of proteins that share functional and structural similarities and are involved in the transport of small hydrophobic molecules such as steroids and lipids into the cell. Lcns are found in many different cell types from plants and bacteria through invertebrate cells to cells of vertebrate origin. The purpose of this review is to summarize the role of Lcns in airway diseases, focusing on allergic and infectious inflammation. In particular, we will summarize the present knowledge about Lipocalin 1 and Lipocalin 2, where exciting new discoveries in the recent years have highlighted their role in pulmonary disease and infection. This new class of proteins is another putative candidate for the development of novel drugs against airway inflammation.

Bβ15–42 Attenuates the Effect of Ischemia-Reperfusion Injury in Renal Transplantation

Renal ischemia-reperfusion contributes to reduced renal allograft survival. The peptide Bβ(15-42), a breakdown product of fibrin, attenuates inflammation induced by ischemia-reperfusion in the heart by competitively blocking the binding of leukocytes to endothelial VE-cadherin, but whether it could improve outcomes in renal transplantation is unknown. Here, we tested the ability of Bβ(15-42) to ameliorate the effects of renal ischemic injury during allogenic kidney transplantation in mice. In our renal transplantation model (C57BL/6 into BALB/c mice), treatment with Bβ(15-42) at the time of allograft reperfusion resulted in significantly improved survival of recipients during the 28-day follow-up (60% versus 10%). Bβ(15-42) treatment decreased leukocyte infiltration, expression of endothelial adhesion molecules, and proinflammatory cytokines. Treatment significantly attenuated allogenic T cell activation and reduced cellular rejection. Moreover, Bβ(15-42) significantly reduced tubular epithelial damage and apoptosis, which we reproduced in vitro. These data suggest that Bβ(15-42) may have therapeutic potential in transplant surgery by protecting grafts from ischemia-reperfusion injury.

Delineating the Role of Histamine-1- and -4-Receptors in a Mouse Model of Th2-Dependent Antigen-Specific Skin Inflammation

Background Histamine drives pruritus in allergic skin diseases which clinically constitutes a most disruptive symptom. Skin pathology in allergic skin diseases is crucially influenced by different T-helper subsets. However, the contribution of different histamine-receptors to T-helper cell dependent skin pathology has not been definitively answered. Models which can specifically address the functional role of T-helper subsets and the mediators involved are therefore valuable to gain further insights into molecular pathways which contribute to allergic skin disease. They might also be helpful to probe amendable therapeutic interventions such as histamine-receptor antagonism. Objective Establishing an adoptive transfer model for antigen-specific Th cells, we aimed to delineate the role of histamine H1- and H4-receptors in Th2-dependent skin inflammation. Methods In-vitro differentiated and OVA primed Th2 cells were adoptively transferred into congenic recipient mice. In vivo treatment with specific histamine H1- and H4-receptor antagonists was performed to analyze the contribution of these histamine-receptors to Th2-dependent skin pathology in our model. Analysis four days after epicutaneous challenge comprised skin histology, flow cytometric detection of transferred T-helper cells and analysis of antigen-cytokine profiles in skin-draining lymph nodes. Results Use of specific H1- and H4-receptor antagonists revealed a crucial role for H1- and H4-receptors for Th2 migration and cytokine secretion in a Th2-driven model of skin inflammation. While H1- and H4-receptor antagonists both reduced Th2 recruitment to the site of challenge, local cytokine responses in skin-draining lymph nodes were only reduced by the combined application of H1- and H4-receptor antagonists and mast cell counts remained altogether unchanged by either H1R-, H4R- or combined antagonism. Conclusion Our model demonstrates a role for H1- and H4-receptors in Th2 cell infiltration and cytokine secretion in allergic skin diseases and suggests further studies to evaluate these findings for therapeutic approaches.

IL-4 Attenuates Pulmonary Epithelial Cell-Mediated Suppression of T Cell Priming

We have previously shown that Th2-polarized airway inflammation facilitates sensitization towards new, protein antigens. In this context, we could demonstrate that IL-4 needs to act on cells of the hematopoetic and the structural compartment in order to facilitate sensitization towards new antigens. We thus aimed to elucidate possible mechanisms of action of IL-4 on structural cells choosing to analyze pulmonary epithelial cells as an important part of the lungs structural system. We used a co-culture system of DC- or APC-dependent in vitro priming of T cells, co-cultivated on a layer of cells of a murine pulmonary epithelial cell line (LA-4) pretreated with or without IL-4. Effects on T cell priming were analyzed via CFSE-dilution and flow cytometric assessment of activation status. Pulmonary epithelial cells suppressed T cell proliferation in vitro but this effect was attenuated by pre-treatment of the epithelial cells with IL-4. Transwell experiments suggest that epithelial-mediated suppression of T cell activation is mostly cell-contact dependent and leads to attenuation in an early naive T cell phenotype. Secretion of soluble factors like TARC, TSLP, GM-CSF and CCL20 by epithelial cells did not change after IL-4 treatment. However, analysis of co-stimulatory expression on pulmonary epithelial cells revealed that pre-treatment of epithelial cells with IL-4 changed expression GITR-L, suggesting a possible mechanism for the effects observed. Our studies provide new insight into the role of IL-4 during the early phases of pulmonary sensitization: The inhibitory activity of pulmonary epithelial cells in homeostasis is reversed in the presence of IL-4, which is secreted in the context of Th2-dominated allergic airway inflammation. This mechanism might serve to explain facilitated sensitization in the clinical context of polysensitization where due to a pre-existing sensitization increased levels of IL-4 in the airways might facilitate T cell priming towards new antigens.

LIPOPOLYSACCHARIDE STIMULATION OF DENDRITIC CELLS INDUCES INTERLEUKIN-10 PRODUCING ALLERGEN-SPECIFIC T CELLS IN VITRO BUT FAILS TO PREVENT ALLERGIC AIRWAY DISEASE

Dendritic cells (DCs) play an important role in directing naive T cells towards a Th1/Th2 or regulatory T cells (Treg) cell phenotype. In this context, interleukin (IL)-10 has been shown to exhibit immune regulatory capacities. The aim of this study was to delineate the influence of high-IL-10–producing DCs on DC–T-cell interactions in inhibiting allergen-induced airway inflammation and hyperreactivity in a murine model of allergic airway disease. Bone marrow–derived dendritic cells (BMDCs) were generated from hemopoietic progenitors by culture with granulocte-macrophage colony-stimulating factor (GM-CSF), and stimulated with ovalbumin (OVA) ± lipopolysaccharide (LPS). The effects of ovalbumin-pulsed BMDCs on cytokine production by allergen-specific naive T cells were studied in vitro. The development of airway inflammation in Balb/c mice was determined after intranasal administration of BMDCs in vivo. LPS stimulation of BMDCs strongly enhanced IL-10 production. Coculture of LPS-modulated DCs exhibiting increased IL-10 production with allergen-specific naive T cells reduced the production of interferon (IFN)-γ and IL-5, but enhanced the production of IL-10. After blockade with anti-IL-10 plus anti-IL-10-receptor antibodies, the level of IFN-γ and IL-5 production by cocultured T cells was restored, underlining the regulatory function of IL-10. Intranasal administration of high-IL-10–producing LPS-stimulated, OVA-primed BMDCs prior to repetitive airway allergen challenges resulted in an even enhanced airway inflammation. These data demonstrate that increased IL-10 production by DCs may be a critical element for T-cell activation and differentiation in the context of allergen-induced immune responses in vitro. However, this DC modulation did not translate into suppression of allergic airway disease in vivo.

Rapid Whole Blood Flow Cytometric Test to Detect ICOS Deficiency in Patients with Common Variable Immunodeficiency

Background/Aims: Common variable immunodeficiency (CVID) is the most common primary immunodeficiency. With respect to underlying defects it comprises a heterogeneous group of deficiencies. For some patients, distinct phenotypical abnormalities have been described, e.g. partial CD40L deficiency or complete ICOS deficiency. For the diagnosis of CD40L deficiency, a rapid whole blood flow cytometric method has been described several years ago. We aimed to determine if the same method can be used to diagnose ICOS deficiency. Methods: Whole blood from 8 healthy volunteers was stimulated for 4 and 20 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Induction of ICOS expression was analyzed on CD8–CD3+ lymphocytes using three-color flow cytometry. Blood from a patient with diagnosed ICOS deficiency was also analyzed. Results: Whole-blood stimulation with PMA and ionomycin for 20 h resulted in a significant induction of ICOS expression on CD8–CD3+ lymphocytes in healthy volunteers. Four-hour incubation also demonstrated ICOS upregulation but to a much lower extent. In CD8–CD3+ lymphocytes from an ICOS-deficient patient, no ICOS expression could be induced following 20 h of stimulation. Conclusion: ICOS expression can be analyzed using a rapid whole blood flow cytometric test.