Francisella tularensis LVS grown in macrophages has reduced ability to stimulate the secretion of inflammatory cytokines by macrophages in vitro

The virulence of Francisella tularensis LVS is determined in part by its ability to invade and replicate within macrophages and stimulate the production of inflammatory cytokines. The present study determined the effects of growing F. tularensis in macrophages on its ability to stimulate cytokine secretion by macrophages. F. tularensis grown in Mueller–Hinton broth (FtB) stimulated the secretion of large amounts of TNF-α, IL-12p40, IL-6 and MCP-1/CCL2 when incubated with macrophages overnight. In contrast, F. tularensis released from infected macrophages (FtMac) stimulated very little secretion of these cytokines by primary cultures of murine peritoneal macrophages, human monocytes or macrophage cell lines. Stimulation of nitric oxide production by FtMac was also less than that elicited by FtB. FtMac killed with gentamicin or paraformaldehyde also stimulated low levels of cytokine secretion. FtMac recovered the ability to stimulate cytokine secretion after overnight culture in broth. Infection of macrophages with FtMac inhibited the cytokine response to subsequent stimulation with LPS from Escherichia coli but did not affect Fcγ receptor-mediated phagocytosis. FtMac were ingested by macrophages at about half the rate of FtB, however, this did not account for the lower cytokine secretion. FtMac and FtB replicated at similar rates within macrophages. Finally, Mice infected with FtMac had a higher mortality rate than those infected with FtB. These results reveal that growth in macrophages causes a reversible phenotypic change in F. tularensis that is associated with decreased stimulation of cytokine secretion, inhibition of LPS-stimulated secretion of inflammatory cytokines by macrophages and increased lethality in mice.