|Ph.D Student||Isakov Elina|
|Subject||Regulation by Nitric Oxide and Thioredoxin of the|
Toll-Like Receptor 4/Interleukin-1B Pathway
|Department||Department of Medicine||Supervisor||Professor Moran Benhar|
|Full Thesis text|
Innate immune cells, including macrophages, express pattern recognition receptors (PRRs) that sense diverse pathogen-associated molecular patterns (PAMPs, such as DNA and lipoproteins) and other “danger signals”. The sensing of PAMPs is largely mediated by Toll-like receptors (TLRs) and Nod-like receptors (NLRs). Several NLRs, including NLRP3, are involved in the assembly of a multiprotein platform called the "inflammasome", which is responsible for caspase-1 activation, leading to processing of key pro-inflammatory cytokines, including IL-1β.
The free radical nitric oxide (NO) plays a central role in innate immunity. Macrophages can generate, via the inducible NO synthase (iNOS), high levels of NO that serve to kill invading pathogens. NO can also regulate macrophage function through protein S-nitrosylation, the covalent attachment of a NO group to the thiol side chain of cysteine. Protein denitrosylation is mediated in part by the thioredoxin (Trx) system, which comprises Trx proteins, Trx reductase (TrxR) proteins and NADPH. However, exactly how nitrosylation/denitrosylation regulate macrophage function is largely unknown.
To investigate how S-nitrosylation and the Trx system regulate TLR/NLR signaling in macrophages we focused on two specific goals: (1) Study of the regulation by NO/S-nitrosylation of the macrophage inflammasome/IL-1β pathway; (2) Investigation of the regulation by Trx system of TLR4-dependent transcriptional response and activation of IL-1β.
Using J774 A.1 macrophages, we found that inhibition of iNOS enhances IL-1β secretion triggered by lipopolysaccharide (LPS) in combination with different agonists of the NLRP3 inflammasome. Co-stimulation with interferon-γ (IFN-γ) potentiates NO generation, and acts upstream of the inflammasome to suppress the induction of pro-IL-1β. However, in conditions of intermediate levels of NO (without IFN-γ), the inhibition of IL-1β secretion appears to occur at the level of the inflammasome, likely via nitrosylation of caspase-1. Inhibition of the TrxR with a potent and specific inhibitor, auranofin, augments nitrosylation of caspase-1 and attenuates secretion of IL-1β.
We then examined how inhibition of TrxR affects TLR4-induced gene activation in macrophages. Global transcriptional profiling and QPCR analyses revealed that treatment with auranofin exerts a selective effect on LPS-induced gene expression, suppressing the induction of a small number of genes, notably IL-1β and NLRP3. Consistently, auranofin suppressed NLRP3 inflammasome-mediated effector functions.
This study shows that NO/S-nitrosylation negatively regulates the TLR4/NLRP3/IL-1β pathway, both upstream to and at the level of the inflammasome. Our findings support a regulatory role for the Trx system in the early phase of TLR4 signaling by positively regulating the transcriptional activation of IL-1β and NLRP3.