Entwicklung und Anwendung eines in vitro Co-Kultur-Modells der Haut zur Identifizierung potentieller Therapeutika bei Schwefellost-Vergiftungen Development and Application of a in vitro Co-Culture Model of the Skin for the Identificaton of Therapeutics against Sulfur Mustard Intoxications

Sulfur mustard (SM) is a vesicant agent that had its first military use 100 years ago, at Ypres. Recently, the use of SM in Syria 2015 was verified and moved the difficulties which are linked with the treatment of SM intoxications back into the spotlight. A SM intoxication especially affects eyes, lung and skin and leads dose dependently to necrosis, apoptosis and inflammation. No causal antidote to counteract SM toxicity exits, so far. Therefore, treatment is mainly symptomatic and in some cases even empiric. Aim of this work was the establishment and testing of a co-culture model of the skin, composed of keratinocytes (HaCaT) and immunocompetent cells (THP-1). This in vitro model reflects the physiology of the skin more closely than a monoculture of keratinocytes and can be used to identify potential candidate substances for the treatment of SM intoxications and evaluation of the current therapy recommendations, respectively. Moreover, it diminishes the need for animal trials. For the validation of the co-cultur-model we used two different strategies. Firstly, we inoculated unexposed THP-1 cells with SM-exposed HaCaT cells. This approach simulates the migration of macrophages from the blood into the inflamed tissue. Our results showed, that healthy THP-1 cells do not aggravate the course of a SM intoxication. On the contrary, the THP-1 cells significantly reduced SM toxicity in the more severely poisoned cells, if the number of THP-1 cells was sufficient. Secondly, adherent HaCaT-cells were inoculated with THP-1 cells before the SM exposure and thus being poisoned simultaneously. The data showed, that a ratio of 2 % THP-1 cells relating to the number of seeded HaCaT cells is sufficient to aggravate necrosis, apoptosis and inflammation in a highly significant manner. In conclusion, the co-culture-model containing HaCaT and THP-1 cells mimics the physiology of the skin in vitro more closely than a HaCaT monoculture. The co-culture model which exposes the HaCaT and THP-1 cells simultaneously to SM has been used in further experiments to ascertain the efficacy of anti-inflammatory drugs against SM intoxication and draw recommendations for SM treatment thereof. For comparability reasons a HaCaT monoculture was treated under the same conditions as the co-culture. In the results, dexamethasone significantly decreased apoptosis and interleukin production when applied in low concentrations. This effect was in the monoculture more clearly than in the co-culture, which led to the conclusion, that the protective effects of dexamethason did not arise from a modulation of immunocompetent cells in first line. Ibuprofen, in the used concentrations, led to a strong increase in SM cytotoxicity in the monoas well as the co-culture. For that reason, ibuprofen cannot be recommended for the treatment of SM intoxications. Diclofenac on the other hand was able to attenuate SM toxicity. The protective effect was stronger in the co-culture compared to the monocultur, which indicates that diclofenac modulates the response of the immunocompetent cells to a SM exposure. The full protective effects of diclofenac could only be carved out by the use of the new established co-culture model while they would have been underestimated in a HaCaT monoculture screening. In summary, the establishment of a valid co-culture–model of the skin was successful. The model was also used to evaluate anti-inflammatory compounds which generated treatment recommendations for the therapy of SM intoxications.