Analyse der Acinetobacter lwoffii F78-induzierten proinflammatorischen Signale und der epigenetischen Veränderungen in CD4+ T-Zellen anhand eines in vitro Zellkulturmodells

In addition to genetic predisposition environmental factors are thought to be responsible for steadily increasing prevalence of asthma bronchiale in industrialized countries in the last decades. The hygiene hypothesis implies that exposure to microbial stimuli prevents from allergic diseases. Recent epidemiological studies have demonstrated a reduction of allergic disease after exposition to microbial components in a farming environment. Subsequently the apathogenic, gram-positive bacterium A. lwoffii F78 was isolated from the stable dust of rural cow sheds [189-191]. Based on these findings, the intranasal exposure of A. lwoffii F78 to Ovalbumin (OVA)-sensitized and -challenged BALB/c-mice significantly reduced the development of an asthmatic phenotype in an animal model of experimental asthma [198]. The aim of the present work was first of all to characterize the effect of exposure to ethanol killed A. lwoffii F78 on the development to the asthmatic phenotype of in an acute mouse model of allergic airway inflammation. In fact, it could be shown that both ethanol killed as well as living intranasal administration of A. lwoffii F78 were able to reduce the asthmatic phenotype significantly in mice. The protective effect of A. lwoffii F78 led to a clear improvement of the airway reactivity in the lung, decreased levels of eosinophilic granulocytes and TH2 cytokine expression in the BAL. The histological analysis of the lungs revealed a highly reduced airway inflammation, reduced number of goblet cells and eosinophilic granulocytes in the airways. Finally, the effectiveness of reproduced phenotypes resulted with development of a protective immune modulatory phenotype induced by A. lwoffii F78, which was not affected in protection by ethanol killed bacteria. It is known that the proinflammatory cytokine response plays an important role in the transmaternal asthma protection, which is mediated by prenatal exposure to A. lwoffii F78 [198]. In our group it has been shown that A. lwoffii F78 exposure to pregnant mother mice led to an activation of the innate immune system accompanied by increased levels of the cytokines IL-6 and TNFα, which are essential for the asthma protection of the progeny [198]. In this study, an in vitro T-cell system was established allowing more detailed investigation of the underlying cellular and molecular mechanisms of the innate and adaptive immune response that play an important role in the mediation of microbial induced asthma protection through A. lwoffi F78 and Cholera Toxin. For this, the first step was the analysis of A. lwoffii induced proinflammatory cytokine profile and kinetic of cells of the innate immune system like murine macrophage culture cells and peritoneal macrophages in vitro. As expected, stimulation with A. lwoffi F78 (living and ethanol killed exposure) led to a dose-dependent increase of proinflammatory cytokines IL-6 and TNFα at the protein level. The treatment with cholera toxin b-subunit (CTB) showed a similar cytokine profile of IL-6 and TNFα, whereas by treatment with the cholera toxin total protein (CT) proinflammatory cytokine expression was not affected. In the next part of the in vitro studies the effects of A. lwoffii F78 stimulated macrophage culture supernatants containing significant levels of expressed IL-6 and TNFα, on naive CD4+ T-cells, were analysed using a new established in vitro T-cell system. By means of these experiments a significant increase of T-cell cytokine production of IL-17A, IL-10 and IL-13 in CD4+ T-cells were detected. The observed mRNA expression reflected approximately indeed levels of the protein expression. By the addition of recombinant proteins (rhIL-6 and rhTGFβ) during the in vitro culturing of naive CD4+ T-cells a significant increase of the cytokines IL-17A, are IL-10 and IL-13 were detected too. Thus, these results allowed drawing the conclusion that the A. lwoffii F78 induced proinflammatory cytokine IL-6 of the innate immune cells (macrophages) is responsible for polarizing and secretion of IL-17A, IL-10 and IL-13 in CD4+ T-cells. However the T-cell cytokine production of IL-17A by culturing of naive CD4+ T-cells with cholera toxin stimulated macrophage culture supernatants, IL-6 plays no pivotal role. In this regard, the IL-21 which is well known for induction of IL-17A expression via alternative signaling pathways, may play a crucial role on the formation of the T-cell response by cholera toxin induced proinflammatory cytokines. Consequently, collected data of these studies redound a completing and extension of the in vitro model of T-cell mediated immune response by microbial stimuli. These data demonstrate for the first time, immunoregulatory effects of the innate immune response by A. lwoffii F78 and cholera toxin were involved to analysis of in vitro cytokine expression by the downstream, adaptive immune response. Thus, besides being known corresponding subpopulations an independent T helper cell subtype of IL-17A and IL-10-producing TH17 cells could be identified by this indirect stimulation with A. lwoffii F78 and cholera toxin-induced cytokines. As already described, mediator-induced interactions between innate and adaptive immunity via microbial stimuli in inflammatory reactions, possessed hypothetically diverse perspectives. Still, it is conceivable that involved proinflammatory cytokines or further mediators of the TH17 signaling pathway antagonize for induction of an anti-allergic, anti-inflammatory signaling pathway (e.g. chemokine responses). In this context, there is discussion about induction of recently discovered TH17 cells by a rural environment may have regulating effect on TH1/TH2 balance by IL-17 secretion [137]. In the last part of this work the intranasal exposure of cholera toxin (CT and CTB) has been analysed in an acute model of allergic airway inflammation to characterize the potential allergoprotective effect of cholera toxin in microbially-mediated asthma protection. The analysis of the asthmatic phenotype revealed a protective effect on the development of experimental asthma in mice by intranasal exposure to CT total protein. This treatment resulted in a significant reduction of TH2 cytokines and the eosinophil granulocytes in the BAL as well as in the tissue. Furthermore, these animals showed a tended improvement of the airway hyper responsiveness. In addition, there was no initiation of an allergen-specific TH1 response (IFNγ) found. However, the analysis of CTB administration revealed elevated levels of eosinophils in both the BAL and the tissue. Therefore protective properties could be excluded for CTB administration in vivo. Additionally, to investigate the relevance of the modified proinflammatory cytokine production, the expression of IL-6 and TNFα was investigated locally and systemically after short term stimulation with CT and CTB in vivo. This in vivo treatment correlated with the results of in vitro experiments regarding levels of BAL cytokine expression. Altogether, there was no induction of an altered locally cytokine secretion of IL-6 and TNFα by short term stimulation with CT. Thus, these data provide the first evidence of a protective potential of cholera toxin (CT) associated with allergic asthma. Furthermore, the results of cholera toxin induced immunomodulatory response by cytokine profile represent a promising approach for examination of IL-6 independent signaling pathways that play a role in the mediation of anti-allergic immune response by cholera toxin. Although the observed effects are not due to an isolated repression of a TH2 by a TH1 immune response, a parallel existing TH17 response through IL-6 independent signaling pathways seems likely. This response is presumably counteracting to an allergen-specific TH2 response and accountable for the anti-allergic, protective effect. The represented mouse model provides new opportunities to examine immunomodulatory effects of cholera toxin in an acute model of allergic airway inflammation and offers new perspectives to the study microbially mediated allergy protection. Furthermore, interesting views on the possible use of cholera toxin in the treatment of allergic asthma in human can now be offered. Currently, bacterial antigens are the focus of new treatment strategies for development of a preventive allergen vaccination to protect against allergic diseases. However, the relevance of these antigens are not isolated single substances. More efficient mix of bacterial cocktails with different microbial components are in consideration. The early interaction of different microbial factors during maturation and programming of the immune system could lead to tolerance formation to develope a successful prevention of allergies and asthma.