Macrophage Proinflammatory Response to Francisella tularensis Live Vaccine Strain Requires Coordination of Multiple Signaling Pathways

Abstract

The macrophage proinflammatory response to Francisella tularensis (Ft) live vaccine strain (LVS) was shown previously to be TLR2 dependent. The observation that intracellular Ft LVS colocalizes with TLR2 and MyD88 inside macrophages suggested that Ft LVS might signal from within the phagosome. Macrophages infected with LVSΔiglC, a Ft LVS mutant that fails to escape from the phagosome, displayed greatly increased expression of a subset of TLR2-dependent, proinflammatory genes (e.g., Tnf) but decreased expression of others (e.g., Ifnb1). This latter subset was similarly mitigated in IFN-β−/− macrophages indicating that while Ft LVS-induced TLR2 signaling is necessary, cytosolic sensing of Ft to induce IFN-β is required for full induction of the macrophage proinflammatory response. Although LVSΔiglC greatly increased IL-1β mRNA in wild-type macrophages, protein secretion was not observed. IL-1β secretion was also diminished in Ft LVS-infected IFN-β−/− macrophages. rIFN-β failed to restore IL-1β secretion in LVSΔiglC-infected macrophages, suggesting that signals in addition to IFN-β are required for assembly of the inflammasome and activation of caspase-1. IFN-β plays a central role in controlling the macrophage bacterial burden: bacterial recovery was greater in IFN-β−/− than in wild-type macrophages and treatment of Ft LVS-infected macrophages with rIFN-β or 5,6-dimethylxanthenone-4-acetic acid, a potent IFN-β inducer, greatly decreased the intracellular Ft LVS burden. In toto, these observations support the hypothesis that the host inflammatory response to Ft LVS is complex and requires engagement of multiple signaling pathways downstream of TLR2 including production of IFN-β via an unknown cytosolic sensor and activation of the inflammasome.

IglG and IglI of the Francisella Pathogenicity Island Are Important Virulence Determinants of Francisella tularensis LVS

“…Thus, both IglG, to some extent, and, even more so, IglI are important for the IL-1␤ release by LVS, consistent with their delayed escape from the phagosomes. Thus, as suggested previously, there is a strong correlation between production of active IL-1␤ protein and a cytoplasmic location of the bacterium (4,21,28,38).…”

“…F. tularensis phagosomal escape into the macrophage cytosol is critical for the inflammasome-dependent induction of IL-1␤ secretion (4, 21, 28, 38, 52). Consequently, macrophages infected with strains with mutations in mglA, iglC, vgrG, or iglI have been shown to display abrogated IL-1␤ release (4,21,28,38,76). To determine whether PdpE, IglG, and IglI play a role in this process, we measured the concentration of IL-1␤ in culture supernatants of macrophages infected with the corresponding LVS mutants at 0, 5, 8, or 24 h postinfection.…”

“…Upon encounter with a host cell, rapid induction of a proinflammatory response which is completely or partly repressed upon bacterial internalization occurs (32,60,74). Moreover, F. tularensis actively suppresses the ability of both dendritic cells and macrophages to secrete cytokines in response to secondary stimuli (8,21,73). Thus, F. tularensis is clearly able to modulate many levels of the host immune response to facilitate its intracellular survival.…”

Phosphatidylinositol 3-Kinase Activation Attenuates the TLR2-Mediated Macrophage Proinflammatory Cytokine Response toFrancisella tularensisLive Vaccine Strain

“…These observations, coupled with the observation that TNF and IL-6 expression are completely dependent on p38 and/or ERK1/2 activity, strongly suggest that the increased cytokine levels in response to inhibition of PI3K result from enhanced and sustained MAPK activation. Importantly, wortmannin treatment did not inhibit bacterial uptake, which could potentially lead to sustained TLR signaling at the macrophage surface or in the phagosome (27). PI3K has also been shown to suppress proinflammatory cytokine production in human monocytes and PBMCs in response to various defined TLR ligands by inhibiting GSK3b activity (48).…”

“…Interestingly, TLR2-dependent proinflammatory cytokine responses in the lung are significantly delayed (7,8), and several in vitro studies have suggested that F. tularensis can directly inhibit proinflammatory signaling in infected macrophages and dendritic cells (7,9,10,26). Although progress has been made in understanding the roles of TLRs and other PRRs (27)(28)(29) in recognizing F. tularensis, little is known about how F. tularensis modulates PRR signaling to its advantage during infection. The PI3K/Akt pathway has emerged as an important regulator of innate immune responses.…”