Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface

The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA

Hypoxia-inducible factor 1α modulates metabolic activity and cytokine release in anti-Aspergillus fumigatus immune responses initiated by human dendritic cells

The mold Aspergillus fumigatus causes life-threatening infections in immunocompromised patients. Over the past decade, new findings in research have improved our understanding of A. fumigatus-host interactions, including the recent identification of myeloid-expressed hypoxia-inducible factor 1α (HIF-1α) as a relevant immune-modulating transcription factor and potential therapeutic target in anti-fungal defense. However, the function of HIF-1α signaling for human anti-A. fumigatusimmunity is still poorly understood, including its role in dendritic cells (DCs), which are important regulators of anti-fungal immunity. This study investigated the functional relevance of HIF-1α in the anti-A. fumigatus immune response initiated by human DCs. Hypoxic cell culture conditions were included because hypoxic microenvironments occur during A. fumigatus infections and may influence the host immune response. HIF-1α was stabilized in DCs following stimulation with A. fumigatus under normoxic and hypoxic conditions. This stabilization was partially dependent on Dectin-1, the major receptor for A. fumigatus on human DCs. Using siRNA-based HIF-1α silencing combined with genome-wide transcriptional analysis, a modulatory effect of HIF-1α on the anti-fungal immune response of human DCs was identified. Specifically, the difference in the transcriptomes of HIF-1α silenced and non-silenced DCs indicated that HIF-1α contributes to DC metabolism and cytokine release in response to A. fumigatus under normoxic as well as hypoxic conditions. This was confirmed by further down-stream analyses that included metabolite analysis and cytokine profiling of a time-course infection experiment. Thereby, this study revealed a so far undescribed functional relevance of HIF-1α in human DC responses against A. fumigatus

Fever-range temperature modulates activation and function of human dendritic cells stimulated with the pathogenic mould Aspergillus fumigatus

In immunocompromised patients, invasive aspergillosis (IA) is the most frequent disease caused by the pathogenic mould Aspergillus fumigatus. Fever is one of the most common yet nonspecific clinical symptoms of IA. To evaluate the role of hyperthermia in the innate immune response to A. fumigatus in vitro, human monocyte-derived dendritic cells (DCs) were stimulated with germ tubes of A. fumigatus or the fungal cell wall component zymosan at 37◦C or 40◦C, followed by characterization of specific DC functions. While maturation of DCs was enhanced and DC phagocytic capacity was reduced at 40◦C, we observed that DC viability and cytokine release were unaffected. Thus, our results suggest that hyperthermia has substantial impacts on DC function in vitro, which might also influence the course and outcome of IA in immunocompromised patients

Genome-Wide Expression Profiling Reveals S100B as Biomarker for Invasive Aspergillosis

Invasive aspergillosis (IA) is a devastating opportunistic infection and its treatment constitutes a considerable burden for the health care system. Immunocompromised patients are at an increased risk for IA, which is mainly caused by the species Aspergillus fumigatus. An early and reliable diagnosis is required to initiate the appropriate antifungal therapy. However, diagnostic sensitivity and accuracy still needs to be improved, which can be achieved at least partly by the definition of new biomarkers. Besides the direct detection of the pathogen by the current diagnostic methods, the analysis of the host response is a promising strategy toward this aim. Following this approach, we sought to identify new biomarkers for IA. For this purpose, we analyzed gene expression profiles of hematological patients and compared profiles of patients suffering from IA with non-IA patients. Based on microarray data, we applied a comprehensive feature selection using a random forest classifier. We identified the transcript coding for the S100 calcium-binding protein B (S100B) as a potential new biomarker for the diagnosis of IA. Considering the expression of this gene, we were able to classify samples from patients with IA with 82.3% sensitivity and 74.6% specificity. Moreover, we validated the expression of S100B in a real-time reverse transcription polymerase chain reaction (RT-PCR) assay and we also found a down-regulation of S100B in A. fumigatus stimulated DCs. An influence on the IL1B and CXCL1 downstream levels was demonstrated by this S100B knockdown. In conclusion, this study covers an effective feature selection revealing a key regulator of the human immune response during IA. S100B may represent an additional diagnostic marker that in combination with the established techniques may improve the accuracy of IA diagnosis

First Insights in NK—DC Cross-Talk and the Importance of Soluble Factors During Infection With Aspergillus fumigatus

Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal

Validation of a simplified in vitro Transwell® model of the alveolar surface to assess host immunity induced by different morphotypes of Aspergillus fumigatus

Interactions between fungal pathogens such as Aspergillus fumigatus with host alveolar epithelium and innate immune cells are crucial in the defense against opportunistic fungal infections. In this study a simplified Transwell® system with a confluent layer of A549 cells acted as a model for the alveolar surface. A. fumigatus and dendritic cells were added to simulate the spatial and cellular complexity in the alveolus. Fungal growth into the lower chamber was validated by galactomannan assays. Addition of moDCs to the upper chamber led to a reduced GM signal and fungal growth, indicating moDC antifungal activity. Minimal cell death was documented by analyses of lactate dehydrogenase concentrations and pro-apoptotic gene expression. Measurement of trans-epithelial dextran blue movement confirmed tightness of the epithelial barrier even in presence of A. fumigatus. Cytokine measurements in supernatants from both chambers of the Transwell® system documented distinct response patterns during early and late stages of epithelial invasion, with A549 cells appearing to make a minimal contribution to cytokine release. Concentrations of cytokines in the lower chamber varied distinctly from the upper chamber, depending on the molecular weight of the cytokines. Low inter-assay variability of fungal biomarkers and cytokines was confirmed, highlighting that in vitro models closely mimicking conditions in the human lung can facilitate reproducible measurement of the dynamics of cytokine release and fungal penetration of host epithelia

Correspondence analysis indicating the factors with the greatest influence on the microarray data.

<p>The triangles represent the A549 cells and these clustered closely even in the presence of <i>A. fu</i>migatus germ tubes. Whereas mDC, represented by squares, clustered close to the A549 data except when germ tubes were added which caused a distinct cluster to be formed. The moDC, represented by circles, formed two distinct clusters one consisting of datasets without fungus. The second cluster for datasets with germ tubes was closer to the mDC dataset with germ tubes. These data indicate that including moDC with the A549 layer leads to greater changes in gene expression compared to the A549 layer than in the absence of exogenous immunostimulators such as <i>A. fumigatus</i>. The data indicate that conidia are not strong immunostimulators of A549 cells. The addition of germ tubes caused similar gene expression responses when mDC or moDC were present.</p

Gene expression of immune-related genes in donor myeloid dendritic cells or monocyte-derived dendritic cells induced by the addition of <i>A. fumigatus</i> germ tubes (after 6 hours incubation).

<p>Expression is expressed as Fold Change (Log₂); ratio of gene expression in DC plus <i>A. fumigatus</i> relative to DC minus <i>A. fumigatus</i>.</p

RT-qPCR validation of differential gene expression observed in microarray experiments measuring cellular interactions involving (a) moDC and (b) mDC.

<p>Gene expression data from the immune gene microarray shown as mean log₂ values of ratios of relative expression determined by RT-qPCR compared to array data. Error bars in both charts indicate the standard error.</p

Gene Expression in A549 epithelial cell layer induced by addition of <i>A. fumigatus</i> germ tubes.

<p>Expression is expressed as Fold change (Log₂); ratio of gene expression in cells plus <i>A. fumigatus</i> relative to cells minus <i>A. fumigatus</i>.</p