Stephan Sudowe

Glucose-independent improvement of vascular dysfunction in experimental sepsis by dipeptidyl-peptidase 4 inhibition

AIMS Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel class of drugs for the treatment of hyperglycaemia. Preliminary evidence suggests that their antioxidant and anti-inflammatory effects may have beneficial effects on the cardiovascular complications of diabetes. In the present study, we investigate in an experimental sepsis model whether linagliptin exerts pleiotropic vascular effects independent of its glucose-lowering properties. METHODS AND RESULTS Linagliptin (83 mg/kg chow for 7 days) was administered in a rat model of lipopolysaccharide (LPS) (10 mg/kg, single i.p. dose/24 h)-induced sepsis. Vascular relaxation, reactive oxygen species (ROS) formation, expression of NADPH oxidase subunits and proinflammatory markers, and white blood cell infiltration in the vasculature were determined. Oxidative burst and adhesion of isolated human neutrophils to endothelial cells were measured in the presence of different DPP-4 inhibitors, and their direct vasodilatory effects (isometric tension in isolated aortic rings) were compared. In vivo linagliptin treatment ameliorated LPS-induced endothelial dysfunction and was associated with reduced formation of vascular, cardiac, and blood ROS, aortic expression of inflammatory genes and NADPH oxidase subunits in addition to reduced aortic infiltration with inflammatory cells. Linagliptin was the most potent inhibitor of oxidative burst in isolated activated human neutrophils and it suppressed their adhesion to activated endothelial cells. Of the inhibitors tested, linagliptin and alogliptin had the most pronounced direct vasodilatory potency. CONCLUSION Linagliptin demonstrated pleiotropic vasodilatory, antioxidant, and anti-inflammatory properties independent of its glucose-lowering properties. These pleiotropic properties are generally not shared by other DPP-4 inhibitors and might translate into cardiovascular benefits in diabetic patients.

Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses

Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV). Biolistic transfection with pFascin initiated a marked type 1 immune response characterized by the occurrence of a large population of IFN-gamma-producing T helper (Th) cells in spleen and draining lymph nodes. Consistently, immunoglobulin production was dominated by IgG2a antibodies. In contrast, the humoral response after repeated administration of pCMV was strongly enhanced and characterized by a type 2-like isotype pattern (IgG1 > IgG2a). Cytokine production analysis in vitro indicated compartmentalization of the immune response, revealing large numbers of IL-4-producing Th cells in the lymph nodes and dominant presence of IFN-gamma-producing Th cells in the spleen. Biolistic transfection with pFascin, like immunization with pCMV, led to potent induction of cytotoxic T cells as was assessed by JAM test. Thus gene gun immunization with plasmids that focus transgene expression and antigen production specifically to DC propagates type 1-biased cellular immune responses.

Allergen-induced IgE-dependent gut inflammation in a human PBMC–engrafted murine model of allergy

BACKGROUND Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. OBJECTIVE In this study we developed a human PBMC-engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. METHODS Nonobese diabetic (NOD)-scid-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. RESULTS Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergen-specific proliferation and cytokine production of human CD4(+) T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. CONCLUSION These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions.

Mast Cells Induce Migration of Dendritic Cells in a Murine Model of Acute Allergic Airway Disease

Background: The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway disease. Following activation, mast cells release a variety of stored or de novo-produced inflammatory mediators, several of them being capable of activating DCs. In this study, the role of mast cells on DC migration from the lungs to the thoracic lymph nodes was investigated in sensitized mice. Methods: Mast cell-deficient mice (KitW-sh/W-sh) and their wild-type counterparts were sensitized intraperitoneally with ovalbumine (OVA) in saline and challenged by a single intranasal administration of OVA labeled with a fluorescent dye (OVA-Alexa). Results: Following challenge, the relative and absolute amount of OVA- Alexa-positive DCs was clearly increased in sensitized wild-type mice compared to nonsensitized mice. In contrast, sensitized KitW-sh/W-sh showed no increase in OVA-Alexa-positive DCs compared to nonsensitized mast cell-deficient animals. In sensitized KitW-sh/W-sh mice reconstituted with bone marrow-derived mast cells (BMMCs), the number of OVA- Alexa-positive DCs was comparable to that in sensitized wild-type animals. However, transfer of allergen-exposed BMMCs to sensitized mice prior to airway challenge augmented airway inflammation similarly in wild-type and mast cell-deficient mice. In line with this, sensitization with allergen-pulsed DCs induced allergic airway disease independently of mast cells. Conclusions: This study shows an interaction between mast cells and DCs following allergen challenge in sensitized hosts. However, the function of mast cells can be bypassed in models utilizing activated allergen-exposed DCs to initiate the development of allergic airway disease.

Uptake and presentation of exogenous antigen and presentation of endogenously produced antigen by skin dendritic cells represent equivalent pathways for the priming of cellular immune responses following biolistic DNA immunization

Gene gun‐mediated biolistic DNA vaccination with β‐galactosidase (βGal)‐encoding plasmid vectors efficiently modulated antigen‐induced immune responses in an animal model of type I allergy, including the inhibition of immunoglobulin E (IgE) production. Here we show that CD4+ as well as CD8+ T cells from mice biolistically transfected with a plasmid encoding βGal under the control of the fascin promoter (pFascin‐βGal) are capable of inhibiting βGal‐specific IgE production after adoptive transfer into naïve recipients. Moreover, suppression of IgE production was dependent on interferon (IFN)‐γ. To analyse the modalities of activation of CD4+ and CD8+ T cells regarding the localization of antigen synthesis following gene gun‐mediated DNA immunization, we used the fascin promoter and the keratin 5 promoter (pK5‐βGal) to direct βGal production mainly to dendritic cells (DCs) and to keratinocytes, respectively. Gene gun‐mediated DNA immunization with each vector induced considerable activation of βGal‐specific CD8+ cytotoxic T cells. Cytokine production by re‐stimulated CD4+ T cells in draining lymph nodes and immunoglobulin isotype profiles in sera of immunized mice indicated that immunization with pFascin‐βGal induced a T helper type 1 (Th1)‐biased immune response, whereas immunization with pK5‐βGal generated a mixed Th1/Th2 immune response. Nevertheless, DNA vaccination with pFascin‐βGal and pK5‐βGal, respectively, efficiently inhibited specific IgE production in the mouse model of type I allergy. In conclusion, our data show that uptake of exogenous antigen produced by keratinocytes and its presentation by untransfected DCs as well as the presentation of antigen synthesized endogenously in DCs represent equivalent pathways for efficient priming of cellular immune responses.

Formalin-Fixed Staphylococcus aureus Particles Prevent Allergic Sensitization in a Murine Model of Type I Allergy

Background: Bacterial infections are supposed to act counterregulatory to the development of allergen-specific Th2 immune responses. We analyzed whether administration of extracellular Staphylococcus aureus inhibited experimental sensitization against allergens. Methods: BALB/c mice were immunized with alum-adsorbed ovalbumin (OVA) together with formalin-fixed Staphylococcus particles. OVA-specific antibody production and cytokine synthesis by spleen cells was analyzed. Airway reactivity and cellular infiltration into the airways was assessed after intranasal challenge of mice with OVA. In addition, the capacity of Staphylococcus particles to modulate cytokine production by bone marrow-derived dendritic cells was analyzedin vitro. Results: Simultaneous application of OVA and Staphylococcus particles very efficiently inhibited production of specific IgE and IgG1 as well as secretion of IL-4 and IL-5 by splenocytes, while enhancing IgG2a formation and production of IFN-γ, indicating a shift from a Th2 response towards a Th1-biased response. This effect was not dependent on the expression of protein A by Staphylococcus. An enhanced frequency or activity of regulatory T cells after administration of Staphylococcus particles was not apparent. Treatment of mice with Staphylococcus particles during the sensitization phase prevented lung inflammation (airway hyperreactivity, eosinophilia) after local challenge with OVA. Culture of bone marrow-derived dendritic cells with Staphylococcus particles induced IL-12p35 and p40 mRNA expression as well as secretion of IL-12p70, and increased production of IL-10 mRNA and protein. Conclusions: Administration of formalin-fixed Staphylococcus particles induced Th1-biased immune responses and prevented allergic sensitization.

Antigen dose-dependent suppression of murine IgE responses is mediated by CD4(-)CD8(-) double-negative T cells.

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization.

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice.

Background: It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). Methods: OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. Results: Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-γ) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. Conclusions: Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.

A novel plasmid DNA electroporation method allows transfection of murine DC.

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4(+) T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.

Non‐eosinophilic Airway Hyper‐reactivity in Mice, Induced by IFN‐γ Producing CD4+ and CD8+ Lung T cells, is Responsive to Steroid Treatment

Non‐eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non‐eosinophilic airway inflammation and airway hyper‐responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen β‐galactosidase (pFascin‐βGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of βGal protein (βGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to βGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4+ T cells into Th2 and Th17 cells, pFascin‐βGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin‐βGal mice revealed that CD4+ and CD8+ cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin‐βGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN‐γ in the bronchoalveolar fluid. Our results suggest that non‐eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin‐βGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non‐eosinophilic asthma that respond to inhaled steroids.