The host innate immune system is a delicate balance of inflammatory mediators that eliminate pathogens and negative regulators that minimize collateral tissue damage and re-establish homeostasis. The type I inter- ferons (IFNs), a closely related family of innate cytokines, represent a key host defense program against viral infections that, in contrast, have a great potential for enhancing pathogenesis in bacterial infections. Specifical- ly, we, and subsequently others, have reported that Toll-like Receptor 4 (TLR4)-mediated expression of IFN-? during in vivo infections by Gram negative pathogens (e.g. Salmonella enteriditis serovar Typhimurium (ST)) leads to enhanced bacterial replication and host mortality. To identify the innate immune signaling pathways that limit IFN-? production during bacterial infections, we employed bioinformatics analyses and identified the prostaglandin E2 (PGE2) receptor, EP4, as a critical regulator of the TLR4-mediated, TRIF signaling pathway and IFN-? induction. Our recently published data has revealed that the PGE2/EP4 signaling axis is, in fact, the feedback mechanism that limits TLR4-dependent IFN-? induction in vitro and in vivo by preventing internaliza- tion of the TLR4/MD2 signaling complex into endosomes where it can recruit the adapter molecules, TRAM and TRIF. Our new preliminary data indicates that, mechanistically, PGE2/EP4 likely acts on the small GTPase Rab11a to block TLR4 trafficking. We additionally present new data that indicates PGE2/EP4 positively regu- lates the TLR4 induced autophagic response directed at TRIF. Based on these preliminary findings, the cen- tral hypothesis of this application is that EP4 signaling both restricts TLR4-TRIF complex assembly on the en- dosome and promotes degradation of active TLR4-TRIF complexes.
In Specific Aim 1, we will test this hypoth- esis by delineating the molecular details of the antagonism between PGE2-EP4 and TLR4-TRIF-mediated sig- naling pathways. We hypothesize that the molecular mechanism of PGE2 antagonism of TLR4/TRIF signaling occurs at two points; 1) the early inhibition of TLR4/TRIF complex assembly by restricting intracellular traffick- ing of TLR4 and signaling adaptors through inhibition of a Rab family GTPase, and 2) the subsequent induction of cargo specific autophagy to terminate TRIF signals.
In Specific Aim 2, we will examine the consequences of this antagonism on the biology of ST infection in an in vivo model. The ultimate goal of this work is to identify host-centric innate signaling pathways that can be manipulated therapeutically to reduce the severity of the inflammatory response to bacterial pathogens.
When host white blood cells, called macrophages, interact with bacteria, a series of signals cause the macrophages to release potent inflammatory substances. One such bacteria is Salmonella Typhimurium, an orally acquired pathogen that most often causes severe gastritis in humans. In an experimental model of bacterial infection using LPS, we have identified two interacting signaling pathways that may be manipulated to lessen the severity of Salmonella infections
