Survival of secondary lethal systemic Francisella LVS challenge depends largely on interferon gamma.

Although survival of primary infection with the live vaccine strain (LVS) of Francisella tularensis depends on interferon gamma (IFN-gamma), the relative importance of IFN-gamma to secondary protective immunity in vivo has not been clearly established. Here we examine the role of IFN-gamma in T cell priming and expression of vaccine-induced protection against lethal intraperitoneal challenge of mice. Large amounts of IFN-gamma were detected between days 3 and 7 in the sera of LVS-immunized mice, while relatively small amounts were found transiently after secondary LVS challenge. Consistent with the production of this cytokine, mice lacking IFN-gamma (gamma interferon knockout, GKO, mice) could not be successfully vaccinated with LVS or an attenuated mglA mutant of F. novicida to withstand secondary Francisella LVS challenge. Further, splenocytes from such primed mice did not adoptively transfer protection to naive GKO recipient mice in vivo, nor control the intramacrophage growth of LVS in vitro. Finally, LVS-immune WT mice depleted of IFN-gamma prior to intraperitoneal challenge survived only the lowest doses of challenge. Thus successful priming of protective LVS-immune T cells, as well as complete expression of protection against Francisella during secondary challenge, depends heavily on IFN-gamma.

NK cells activated in vivo by bacterial DNA control the intracellular growth of Francisella tularensis LVS.

We demonstrated previously that mice treated with bacterial or oligonucleotide DNA containing unmethylated CpG motifs are transiently protected against lethal parenteral challenge with the intracellular bacterium Francisella tularensis Live Vaccine Strain (LVS). Here we explore the cellular basis of this protection. Wild-type mice that were treated with CpG oligonucleotide DNA and challenged with a lethal dose of LVS survived, while mice lacking TLR9 did not. In vitro, treatment of LVS-infected macrophages and/or naive splenocytes with oligo DNA had no impact on intracellular bacterial replication. In contrast, in vitro co-culture of LVS-infected macrophages with splenocytes obtained from mice treated with oligo DNA in vivo resulted in control of intracellular LVS growth. Control was reversed by antibodies to interferon-gamma or to tumor necrosis factor-alpha and by inhibition of nitric oxide, and to a lesser degree by antibodies to Interleukin-12. Further, splenocytes from DNA-primed normal, T cell KO, B cell KO, lymphocyte-deficient scid, or perforin KO mice all controlled intra-macrophage LVS growth. Enriched DNA-primed natural killer cells, but not B cells, clearly controlled intracellular LVS growth. Thus, NK cells contribute to DNA-mediated protection by production of cytokines including IFN-gamma and TNF-alpha, resulting in nitric oxide production and control of intracellular Francisella replication.

In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70.

To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-gamma), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4(+) and CD8(+) T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-gamma than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-gamma production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.