Glucocorticoids Differentially Target SOCS1 and Type 1 Interferons to Regulate TLR-Induced STAT-1 Activation.
Authors: Bhattacharyya Sandip , Muglia Louis 
Published: October 2019

Abstract
Abstract 239
Glucocorticoids (GCs) released endogenously or administered clinically limit the severity of a wide range of inflammatory and autoimmune diseases. Understanding GC action in modulating inflammatory reactions holds promise for the development of more efficacious therapy by minimizing the detrimental sequel of these drugs. In the context of inflammatory diseases, Toll-like receptors (TLRs) are well characterized to integrate and propagate inflammatory signals, while GCs inhibit inflammatory gene expression including cytokines. Several cytokines functions via the JAK-STAT (Janus Kinase-Signal Transducer and Activator of Transcription) pathway and promote transcription of STAT-responsive genes. Interestingly, almost all members of mammalian STAT super family, except STAT-1, are known to functionally synergize with GC. In the light of relatively less known GC-antagonizing nature of STAT-1, we explored STAT-1 regulation by GC in macrophages during activation of inflammatory pathways with TLR-specific ligands. We observed selective impairment of STAT-1 activation by GC, depending on the nature of TLR / TLR-adapter engagement. Most TLRs utilize the adapter MyD88 with the exception of TLR3, which exclusively recruits the adapter Trif. TLR4 is the only member that exploits both MyD88 and Trif to induce downstream targets of the signaling cascade. Secretion of proinflammatory cytokines e.g. TNF-α and IL-12 was 50 – 62 % inhibited in STAT-null macrophages during TLR4 (MyD88 / Trif) or TLR3 (Trif) engagement with LPS and Poly (I:C) respectively but not for TLR9 (MyD88) engagement with CpG. We also observed 2 – 15 fold suppression of STAT-1 phosphorylation (at Ser727 and Tyr701) by Dex (Dexamethasone, a chemical analogue of GC) in LPS or Poly (I:C) treated macrophages. There was little effect of Dex during CpG treatment. Pre-treatment with anti-IFN receptor 1 (IFNAR1) antibody indicated TLR3 or TLR4 induced STAT-1 phosphorylation is IFN-dependent. Unexpectedly, IFN-β dependent STAT-1 phosphorylation was found to be Dex-resistant indicating little or no role of GC on IFN-mediated STAT-1 activation. But, we observed 2.5 - 4 fold suppression by Dex for Poly (I:C) induced IFN-α, IFN-β secretion and 2 - 2.8 fold suppression of nuclear interferon regulatory factor 3 (IRF-3) level, but not for LPS. IRF-3 is known as a critical component for IFN induction. Our results indicate GC impairs Trif-mediated STAT-1 activity by inhibiting IFN production and not via direct inhibition of IFN function. CpG did not induce IFN-α, IFN-β secretion or STAT-1 phosphorylation at Tyr701. Furthermore, we found 2 – 5 fold elevation of suppressor of cytokine synthesis 1 (SOCS1) level by Dex in either case of TLR4 or TLR3 engagement. We hypothesize that GC attenuates MyD88–Trif-mediated STAT-1 activation via induction of SOCS1. Intriguingly, inhibition of Trif-mediated STAT-1 activation occurs in two steps; 1) by impairing IFN secretion and 2) by induction of SOCS1. Taken together, our data implicate SOCS1 and type 1 interferons are novel target of GC in regulating STAT-1 activation for TLR-mediated inflammatory responses.

Disclosures:
Muglia: Pfizer Inc.: Research Funding.