Modulation of macrophage proinflammatory functions by cytokine-expressing Salmonella vectors

We previously reported that the intraperitoneal administration of recombinant strains of Salmonella enterica serovar Typhimurium, engineered to express murine IL-2 (designated GIDIL2) or IFN-γ (GIDIFNγ), induced a cytokine-specific modulation of the host innate immune response. Interestingly, the bacteria-expressed cytokines were not secreted, but instead were associated with the bacterial cytosol. To understand the mechanism by which these two transfectants influence immune cells, we investigated their effect on two macrophage populations, J774A.1 cell line and ex vivo isolated peritoneal macrophages (PM). The parental, cytokine-negative, Salmonella strain (designated BRD509E), was used as a control. The capacity of the bacterial strains to activate macrophages was assessed by modulation of surface expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) and activation marker Ly-6A/E, and by induction of cytokine production. Our data revealed that GIDIFNγ was the only strain capable of upregulating the expression of cell-surface markers. Moreover, infection of macrophages with GIDIFNγ induced a stronger cytokine response in comparison with BRD509E or GIDIL2 strain, as demonstrated by the production of TNF-α, IL-6, IL-12/IL23p40 and NO. The ability of GIDIL2 and GIDIFNγ strains to activate macrophages was not due to enhanced invasiveness, as their cellular invasion rates were 2-fold lower than the parental strain. Further investigation of cytokine expression by GIDIL2 and GIDIFNγ strains showed that while the cytokines were not secreted, they were expressed on the bacterial surface suggesting that their effect on macrophages could be through a direct interaction with their receptors on target cells. This was confirmed by showing that cytochalasin D-treated macrophages, a treatment which effectively inhibited bacterial invasion, could be induced to secrete high levels of cytokines by GIDIFNγ organisms. Our data demonstrate that cytokine-expressing bacteria modulate macrophage activation independently of their entry into cells and may explain the rapid action of these bacterial strains when injected systemically into susceptible mice.