IFNαβ, NF-κB, TLR, TNFα, S1P: The αβγs of Endogenous Ligands
CITATION Guiducci C, Gong M, Xu Z, et al. TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus. Nature 2010; 465: 937–941.
CITATION Alvarez SE, Harikumar KB, Hait NC, et al. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 2010; 465: 1084–1088.
CITATION Saini V, Marchese A, Majetschak M. CXC chemokine receptor 4 is a cell surface receptor for extracellular ubiquitin. J Bio Chem 2010; 285: 15566–15576.
SUMMARY AND ANALYSIS
Steroids have remained a mainstay for the treatment and prophylaxis of organ transplant rejection, yet they lack efficacy in treating so-called steroid-resistant rejections. Similarly, many transplant candidates have progressed to end-stage organ failure through the failure of steroids or other immunosuppressive drugs targeting established adaptive immune system pathways. Indeed, the de novo occurrence of autoimmunity post transplant is well documented. Thus, transplant patients continue to suffer from allo- and auto-immune mechanisms with apparent resistance to the inhibition of conventional adaptive immune targets. Several reports now suggest numerous novel targets among endogenous ligands, inflammatory receptors, and the innate immune system with promise to meet this unmet therapeutic niche.
Steroid Resistance and TLRs
Guiducci et al. examined the activity and responses of dendritic cells (DC) in both mice and humans with rheumatoid arthritis (RA). They noted that the subset of plasmacytoid DC (pDC) was activated in RA, and produced large quantities of type I interferon (IFN), a defining characteristic of the pDC subset. The pDC activation was dependent on the nuclear factor (NF)-κB pathway. NF-κB activation was in turn dependent on signaling through Toll-like receptors (TLRs). In particular, TLR7 and TLR9 were stimulated by endogenous nucleic acids and immune complexes. TLR pathway stimulation was insensitive to the inhibitory effects of steroids, and thus pDC continued to produce type I IFN despite administration of steroids. Furthermore, NF-κB activation was a survival pathway, and prevented steroid-induced pDC apoptosis. Combined inhibition of TLR7 and TLR9 resulted in decreased IFN and decreased pDC survival. The implications of these findings are that they highlight targets, which circumvent steroid resistance. The targets (pDCs and TLRs) are components of innate, not adaptive, immunity. Since the ligands for TLR (nucleic acids, immune complexes and ribonucleoproteins) are ubiquitous and endogenous, they are not likely to be modifiable by simple drug therapies. Thus, the observations here guide us toward treatments that specifically focus on the receptors and the cells rather than the ligands.
Many Targets for S1P
Alvarez et al. investigated NF-κB in a model of inflammation induced by tumor necrosis factor α (TNFα). NF-κB activation requires TNF receptor-associated factor 2 (TRAF2), which is a ubiquitin ligase. In a cascade of interactions, TRAF2 ligase activity is associated with polyubiquitination of receptor-interacting protein 1 (RIP1), which then recruits and stimulates inhibitor of κB (IκB) kinase, leading to phosphorylation and dissociation of IκB subunits, and consequent activation of NF-κB. The authors for the first time have provided direct evidence that TRAF2 catalyzes the ubiquitination of RIP1. Unexpectedly, the co-factor for the enzymatic activity was sphingosine 1-phosphate (S1P). S1P is a ubiquitous intracellular and extracellular lipid signaling molecule. It is produced by the action of sphingosine kinase 1 (SphK1) or SphK2. S1P is important in both alloimmunity and autoimmunity, since it activates S1P receptors (S1PRs), which are the targets of the immunosuppressive molecule FTY720. FTY720 (fingolimod, Gilenva, Novartis) is currently FDA approved for treatment of multiple sclerosis. While FTY720 failed to show superiority in kidney transplant trials, it and related compounds are currently undergoing clinical evaluation for other auto- and alloimmune indications. Several interesting implications arise from the study observations. First, does FTY720 bind to or regulate TRAF2? The data presented remain imprecise but suggestive on this point. Perhaps an S1P analogue that blocks both S1PR and TRAF2 will be a more potent and efficacious immunosuppressive. Second, since FTY720 blocks S1PR, will it divert S1P to TRAF2, thereby activating TRAF2 and impeding its own anti-inflammatory effects? Third, the data reveal an endogenous mediator for NF-κB activation, S1P, that may be targeted through a variety of receptors and substrates, including S1PR, SphK1/2, sphingosine lyase (which hydrolyzes S1P) and TRAF2. As noted above, it is not the endogenous ligand, but rather receptors and other substrates that should be targeted for drug development.
Saini et al. studied another aspect of ubiquitin biochemistry. As noted above, ubiquitin is a “ubiquitous” intracellular molecule involved in many signaling pathways, including NF-κB activation. These authors noted that ubiquitin is also found in abundance in the plasma, derived at least in part from damaged erythrocytes and other injured cells. The authors cited reports showing that plasma ubiquitin has anti-inflammatory properties. They demonstrated that ubiquitin bound and activated the chemokine receptor CXCR4 causing cell migration. It should be noted that CXCR4 is one of the two major chemokine receptors for HIV, and is also a major receptor for migration of breast cancer and other neoplastic cells. The investigators related the chemotactic activity of ubiquitin to its anti-inflammatory and immunosuppressive activities, suggesting that it diverted or altered the migration and function of leukocytes. The important implications of this report are that, like S1P—a common molecule distributed widely in intracellular and extracellular compartments—ubiquitin has diverse receptors and functions in each locale, is a component of innate immunity, and as an endogenous and ubiquitous ligand cannot be directly targeted for therapeutic purposes.
Together these reports demonstrate a complex intertwining of multiple signaling pathways of the innate immunity that determine profound activities of inflammation and drug efficacy. They resolve a number of longstanding quagmires about drug resistance and off-target effects. Steroid resistance may not involve escape of the adaptive immune system, but rather resistance of innate immunity. The side effects or lack of efficacy of S1PR modulators may be due to overlap with other previously un-considered activities of S1P. The reports suggest new targets for immune manipulation, and show that endogenous ligands have powerful influences on immune regulation. The challenge will be to target what appear to be very abundant receptors and ligands in a restricted fashion.