Membrane prohibitin forms a dynamic complex with p56lck to regulate T cell receptor signaling.

Prohibitin is a highly conserved ubiquitously expressed protein involved in several key cellular functions. Targeting of this protein in the membrane by the virulence polysaccharide, Vi, of human typhoid-causing pathogen, Salmonella enterica serovar Typhi (S. Typhi), results in suppression of IL-2 secretion from T cells activated through the T-cell receptor (TCR). However, the mechanism of this suppression remains unclear. Here, using Vi as a probe, we show that membrane prohibitin associates with the src-tyrosine kinase, p56lck (Lck), and actin in human model T cell line, Jurkat. Activation with anti-CD3 antibody brings about dissociation of this complex, which coincides with downstream ERK activation. The trimolecular complex reappears towards culmination of proximal TCR signaling. Engagement of cells with Vi prevents TCR-triggered activation of Lck and ERK by inhibiting dissociation of the former from prohibitin. These findings suggest a regulatory role for membrane prohibitin in Lck activation and TCR signaling.

ATP Release Drives Inflammation with Lysophosphatidylcholine.

Lysophosphatidylcholine (LPC), a dominant lipid component of oxidized low-density lipoprotein, plays a major role in inflammation associated with atherosclerosis and neurodegenerative disorders. It activates inflammatory responses from macrophages, neuronal cells, and endothelial cells. However, the exact mechanism by which LPC promotes inflammation remains incompletely understood. In this study, we show that the production of inflammatory cytokines and cytotoxicity with LPC are both critically dependent on its ability to bring about release of ATP from cells. The induction of caspase-1-mediated IL-1ß release with LPC from TLR-primed mouse and human macrophages and mouse neuronal cells is reduced in the presence of ATP-hydrolyzing enzyme, apyrase, and the inhibitors of purinergic signaling. ATP released from LPC-treated cells also promotes an IL-12p70hi, low phagocytic, and poorly costimulatory phenotype in macrophages in a caspase-1-independent manner. Treatment with apyrase reduces production of inflammatory cytokines with LPC in vivo. These findings reveal a previously unappreciated pathway for the generation of inflammatory responses with LPC, and these have significant implications for therapeutic intervention in chronic inflammatory disorders promoted by this lipid.

Serum-borne lipids amplify TLR-activated inflammatory responses.

TLRs recognize conserved pathogen associated molecular patterns and generate innate immune responses. Several circulating and cell membrane associated proteins have been shown to collaborate with TLRs in sensing microbial ligands and promoting inflammatory responses. Here, we show that serum and serum-borne lipids including lysophosphatidylcholine (LPC) amplify inflammatory responses from intestinal epithelial cells and mononuclear phagocytes primed with microbial TLR ligands. Treatment with the inhibitors of G protein-coupled receptor (GPCR) signaling, suramin, or pertussis toxin (PT), the inhibitor of JNK-MAPK, or knockdown of LPC response-regulating GPCR, G2A, decreases the augmentation brought about by serum or LPC in TLR-induced inflammatory response. In vivo administration of PT or anti-G2A antibody reduces TLR2-activated cytokine secretion. The ability of host lipids to costimulate TLR-generated cellular responses represents a novel pathway for the amplification of innate immunity and inflammation.

Lysophosphatidylcholine Potentiates Antibacterial Activity of Polymyxin B.

Polymyxin B, used to treat infections caused by antibiotic-resistant Gram-negative bacteria, produces nephrotoxicity at its current dosage. We show that a combination of nonbactericidal concentration of this drug and lysophosphatidylcholine (LPC) potently inhibits growth of Salmonella and at least two other Gram-negative bacteria in vitro This combination makes bacterial membrane porous and causes degradation of DnaK, the regulator of protein folding. Polymyxin B-LPC combination may be an effective and safer regimen against drug-resistant bacteria.

Innate Activation of IFN-γ-iNOS Axis During Infection With Salmonella Represses the Ability of T Cells to Produce IL-2.

Pathogenic Salmonella serovars are a major cause of enteric illness in humans and animals, and produce clinical manifestations ranging from localized gastroenteritis to systemic disease. T cells are a critical component of immunity against this intracellular pathogen. The mechanisms by which Salmonella modulates T-cell-mediated immune responses in order to establish systemic infection are not completely understood. We show that infection of mice with Salmonella enterica serovar Typhimurium (S. Typhimurium) suppresses IL-2 and increases IFN-γ and IL-17 production from T cells activated in vivo or ex vivo through the T cell receptor. Infection with S. Typhimurium brings about recruitment of CD11b+Gr1+ suppressor cells to the spleen. Ex vivo depletion of these cells restores the ability of activated T cells to produce IL-2 and brings secretion of IFN-γ and IL-17 from these cells back to basal levels. The reduction in IL-2 secretion is not seen in IFN-γ-/- and iNOS-/- mice infected with Salmonella. Our findings demonstrate that sustained innate activated IFN-γ production during progression of infection with Salmonella reduces IL-2-secreting capability of T cells through an iNOS-mediated signaling pathway that can adversely affect long term immunity against this pathogen.