Download In vitro study of host

In vitro study of host-microbe interaction:
co-culture of P. aeruginosa and human cell lines
Mihaela Perić, Mario Matijašić, Hana Čipčić Paljetak and Donatella Verbanac
University of Zagreb School of Medicine,
Center for Translational and Clinical Research, Šalata 2, 10 000 Zagreb, Croatia
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 coculture 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.