<p> In vitro study of host-microbe interaction: co-culture of P. aeruginosa and human cell lines</p><p>Mihaela Perić, Mario Matijašić, Hana Čipčić Paljetak and Donatella Verbanac</p><p>University of Zagreb School of Medicine, Center for Translational and Clinical Research, Šalata 2, 10 000 Zagreb, Croatia</p><p>Host-microbe interactions are important field of research aiming to elucidate signaling and its role in health and disease. Our research was designed to study the signaling involving various human cell lines and human opportunistic Gram-negative bacterial pathogen P. aeruginosa that causes wide spectrum of human infections. Host responds to bacteria first by activating its innate defense system, primary epithelial surfaces and phagocytes. We envisaged an in vitro model where the co-cultivation of more than two communicating partners is involved. Preliminary experiments tested the short termed effects (1 hour) of P. aeruginosa supernatants and cells on monocyte (THP-1) cell line and normal human bronchial epithelia (NHBE) cytokine production separately. Bacterial supernatant did not significantly induce cytokine production in either cell line. On the other hand, bacterial cells in 1.5x105 count and above, increased levels of TNFα and IL-6 in THP-1 and IL-6, IL-8, GM-CSF and MCP-1 in NHBE cells. Bacterial cells in counts above 3x105 were found to impair cell integrity. The subsequent experiments tested overnight influence of P. aeruginosa on NHBE air-liquid co-culture with THP-1 cells. The same set of cytokines was stimulated as in bipartite NHBE culture but the presence of THP-1 cell line in the co- culture contributed to the lower measured concentration of IL-6, IL-8, GM-CSF or MCP-1 after 8 hours of incubation, indicating that macrophages are somehow depleting these cytokines from the culture media. This effect was completely reversed after 24 hours. TNFα was not detected in culture media suggesting that bacteria failed to reach THP-1 cells in lower compartment and the cytokine cocktail produced by NHBE was not able to stimulate THP-1 into producing TNFα. We conclude that multimember in vitro system better describes the dynamic in vivo interactions and presents an attempt to establish a research platform for studying host-pathogen interactions.</p>