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Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.
Solid organ transplantation is an established therapeutic approach in modern medicine to extend and to improve the life of patients in the final stages of organ failure. Transplantation between genetically non-identical individuals leads to the activation of the transplant recipient's immune system. This alloimmune response is a consequence of the recognition of foreign MHC molecules by alloreactive host T cells. To prevent their activation and the subsequently induced activation of further cell subsets (e.g. B cells, cytotoxic T cells, macrophages)immunosuppressive drugs are absolutely necessary in the clinic. However,permanent immunosuppression leads to severe side effects such as nephrotoxicity, diabetes and hyperlipidaemia, and a reduced immunity to infections and malignant diseases. At the moment, there is no real alternative to immunosuppression. The purpose of this study was to analyse the importance of rat dendritic cells with immune inhibitory properties to prevent the immune activation after experimental transplantation. The rat is one of the most important animal models for experimental organ transplantation in a clinic-relevant procedure. In order to modulate the immune response after transplantation in an antigenspecific manner, the strategy should include the alloantigens. These antigens have to be presented by immature dendritic cells in the absence of costimulatory signals in order to turn alloreactive T cells into anergic or regulatory T cells instead of effector T cells. For a certain rat model of allograft rejection,the immunodominant peptide P1 was identified as an important alloantigen which accelerates graft rejection. Such a model offers an attractive and practical approach to analyse the potential of host tolerogeneic dendritic cells pulsed with P1 to suppress the allograft-induced immune response in an antigen-specific manner without the need of chronic immunosuppression. A homogenous population of rat immature dendritic cells was generated from bone marrow precursors cultured with GM-CSF and IL-4 (= IL-4 DCs) or GM65 CSF and IL-10 (= IL-10 DCs). These cells with an identical immature phenotype showed no or a very low surface expression of costimulatory molecules like CD80 and CD86 and a 10-fold reduced expression of MHC class II molecules in comparison to mature splenic DCs. No obvious difference was observed between the phenotype of the IL-4 DCs and the IL-10 DCs. Neither IL-4 DCs nor IL-10 DCs were able to activate naïve T cells or to restimulate antigen-specific T cells. This strong inhibitory effect, mediated within 24 hours, was dependent on the number of immature dendritic cells added to the proliferation assay. Antigen-specific T cells pre-incubated with IL-4 DCs and IL-10 DCs, respectively, were not able to proliferate in the presence of P1-pulsed mature DCs. This anergic state was reversible with the addition of exogenous IL-2. T cells incubated with IL-4 DCs (= IL-4 DC-Ts) were able to inhibit the T cell proliferation in a cell number dependent manner. In contrast, antigen-specific T cells pre-incubated with P1-pulsed IL-10 DCs (= IL-10 DC-Ts)showed no effect on the proliferation assay. This was the unique difference between IL-4 DCs and IL-10 DCs found in the present study. Immature DCs influenced also the immune response after transplantation. Different numbers of P1-loaded immature IL-4 DCs and IL-10 DCs were transferred intravenously into Lewis rats one day before transplantation. The best results were obtained with 30 million P1-pulsed immature DCs which prolonged the survival time to a median of 11.2 ± 1.6 days. In addition, the antigen specificity of this effect was demonstrated with a third-party graft from Brown Norway donors. These findings suggest that an antigen-specific modulation of the immune response is possible using immature dendritic cells loaded with the allogeneic antigens. Even more, the protocols described in the present study show that the immune system can be, at least temporarily, controlled after transplantation without the use of immunosuppressive drugs.