Abstract
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.
Highlights
Invasive mycoses are responsible for significant morbidity and mortality in immuno-compromised patients
PBMC Isolation and Generation of Monocyte-Derived Dendritic Cells Peripheral blood mononuclear cells (PBMC) were obtained by ficoll gradient centrifugation of leukocyte reduction systems kindly provided by the Institute for Transfusion Medicine and Immunohematology Wuerzburg
Spores were diluted at a concentration of 5 × 106 cells per ml in endothelial cell basal medium 2 (EBM2) + 10% fetal calf serum (FCS) + endothelial cell growth medium 2 SingleQuotsTM without gentamicin/amphotericin B (GA-1000), hereinafter called “human pulmonary artery endothelial cell (HPAEC) medium.”
Summary
Invasive mycoses are responsible for significant morbidity and mortality in immuno-compromised patients. Humans are exposed to thousands of airborne mold spores every day and epithelial barriers of the respiratory tract, especially the 0.5–2 μm thin alveolar-capillary barrier with its over 100 m2 surface, commonly represent the primary site of interaction (Croft et al, 2016). In addition to their anatomical barrier function, airway epithelia demonstrably contribute to a fined-tuned immunological balance, which is required to control fungal invasiveness while preventing hypersensitivity, hyperinflammation, and excessive tissue damage (Park and Mehrad, 2009). Infected airway epithelial cells release a broad array of antimicrobial peptides, enzymes, and cytokines, and thereby contribute to both fungal clearance and orchestration of their micro-environment through cytokine signaling (Park and Mehrad, 2009; Osherov, 2012)
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