Co-infection with Staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model

Abstract

Pneumonia is one of the most common infectious diseases worldwide. The influenza virus can cause severe epidemics, which results in significant morbidity and mortality. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus represents a frequent causative bacterial pathogen. Currently available models have several limitations in the analysis of the pathogenesis of infections, e.g. some bacterial toxins strongly act in a species-specific manner. Human 2D mono-cell culture models often fail to maintain the differentiation of alveolus-specific functions. A detailed investigation of the underlying pathogenesis mechanisms requires a physiological interaction of alveolus-specific cell types. The aim of the present work was to establish a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions. We demonstrate that high barrier integrity maintained for up to 14 d in our model containing functional tissue-resident macrophages. We show that flow conditions and the presence of macrophages increased the barrier function. The infection of epithelial cells induced a high inflammatory response that spread to the endothelium. Although the integrity of the epithelium was not compromised by a single infection or co-infection, we demonstrated significant endothelial cell damage associated with loss of barrier function. We established a novel immune-responsive model that reflects the complex crosstalk between pathogens and host. The in vitro model allows for the monitoring of spatiotemporal spreading of the pathogens and the characterization of morphological and functional alterations attributed to infection. The alveolus-on-a-chip represents a promising platform for mechanistic studies of host-pathogen interactions and the identification of molecular and cellular targets of novel treatment strategies in pneumonia.

Staphylococcus aureus Lung Infection Results in Down-Regulation of Surfactant Protein-A Mainly Caused by Pro-Inflammatory Macrophages

“…(ii) In the murine model it is difficult to attribute the host response to a defined cell type. To counter these disadvantages, we recently published a human-alveolus-on-a-chip model that is composed of defined cell types (epithelial, endothelial and immune cells) of human origin [25].…”

“…For the cell cultivation, we use NCI-H441 cells, endothelial cells (human umbilical vein endothelial cells, HUVECs) and human monocyte-derived macrophages (hMdM) as described above cm 2 [25].…”

“…HUVECs were kindly provided by Regine Heller (Faculty of Biological Sciences, Friedrich Schiller University of Jena) and were isolated from human umbilical cord veins as described previously and seeded into the biochip (vascular cavity) at a density of 2.7 × 10 5 HUVECs/cm 2 in endothelial cell growth medium (PromoCell, Heidelberg, Germany) [26] for 48 h with a daily medium exchange [25].…”

“…and transferred to an antibody-coated 96-well plate in duplicates (Human TNF-α ELISA Kit, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and subsequently analyzed according to the manufacturer's instructions. For the identification of SP-A, we used NCI-H441-cells and performed the infection as previously described [25]. Next, 4 h and 18 h p.i.…”

“…After deconvolution with Huygens Software (Scientific Volume Imaging b.v.Hilversum, The Netherlands), the images were segmented in Imaris 9.2.1 (Bitplane, Zürich, Switzerland). The complete process was published before [25].…”