Octyl gallate has potent anti-inflammasome activity by directly binding to NLRP3 LRR domain.

The NOD-, LRR-, and Pyrin domain-containing protein 3 (NLRP3) inflammasome plays key roles in regulating inflammation. Numerous studies show that the abnormal activation of NLRP3 associates with the initiation and progression of various diseases. Hence, the NLRP3 inflammasome may be a promising therapeutic target for these diseases. Octyl gallate (OG) is a small molecule with antioxidant, antimicrobial, antifungal, and anti-inflammatory activities; however, the mechanism underlying its anti-inflammatory activity is still unclear. Here, we developed a screening system for NLRP3-inflammasome inhibitors. A total of 3287 small molecules were screened for inhibitors of nigericin-induced NLRP3 oligomerization. OG was identified as a novel inhibitor. We show that OG directly targets the LRR domain of NLRP3 and thereby blocks the inflammatory cascade of the NLRP3 inflammasome. This contrasts with the mode-of-action of other direct NLRP3 inhibitors, which all bind to the NACHT domain of NLRP3. Interestingly, OG also inhibits the priming step by downregulating the Raf-MEK1/2-ERK1/2 axis. Thus, OG inhibits the NLRP3 inflammasome by two distinct mechanisms. Importantly, OG injection ameliorated the inflammation in mouse models of foot gout and sepsis. Our study identifies OG as a potential therapeutic agent for NLRP3-associated diseases.

Differential Expression Patterns of Toll-like Receptors in COVID-19 Patients.

Since Toll-like receptors (TLRs) recognize the earliest signs of infection or cell damage, they play fundamental roles in innate immunity. This review summarizes the numerous studies on the expression of TLRs in patients with Coronavirus disease 2019 (COVID-19). We show that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can stimulate at least six of the ten TLRs in humans and that this can shape the severity of COVID-19. Specifically, TLR2, TLR4, and TLR9 appear to play pathogenic roles while TLR3, TLR7, and TLR8 may be protective. Most have mutations that could partly explain the susceptibility phenotypes of COVID-19. Further understanding the roles of TLRs in COVID-19 immunopathogenesis could reveal prognostic biomarkers and help drive the development of novel and effective therapeutics for COVID-19.

Pim1 promotes IFN-ß production by interacting with IRF3.

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-ß by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-ß and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-ß signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1-/- mice produced less serum IFN-ß and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-ß production, thus revealing a novel target for controlling antiviral innate immune responses.

Modelling the impact of rapid diagnostic tests on Plasmodium vivax malaria in South Korea: a cost-benefit analysis.

BACKGROUND: Rapid diagnostic tests (RDTs) are widely used for diagnosing Plasmodium vivax malaria, especially in resource-limited countries. However, the impact of RDTs on P. vivax malaria incidence and national medical costs has not been evaluated. We assessed the impact of RDT implementation on P. vivax malaria incidence and overall medical expenditures in South Korea and performed a cost-benefit analysis from the payer's perspective. METHODS: We developed a dynamic compartmental model for P. vivax malaria transmission in South Korea using delay differential equations. Long latency and seasonality were incorporated into the model, which was calibrated to civilian malaria incidences during 2014-2018. We then estimated averted malaria cases and total medical costs from two diagnostic scenarios: microscopy only and both microscopy and RDTs. Medical costs were extracted based on data from a hospital in an at-risk area for P. vivax malaria and were validated using Health Insurance Review and Assessment Service data. We conducted a cost-benefit analysis of RDTs using the incremental benefit:cost ratio (IBCR) considering only medical costs and performed a probabilistic sensitivity analysis to reflect the uncertainties of model parameters, costs and benefits. RESULTS: The results showed that 55.3% of new P. vivax malaria cases were averted, and $696 214 in medical costs was saved over 10 years after RDT introduction. The estimated IBCR was 2.5, indicating that RDT implementation was beneficial, compared with microscopy alone. The IBCR was sensitive to the diagnosis time reduction, infectious period and short latency period, and provided beneficial results in a benefit over $10.6 or RDT cost under $39.7. CONCLUSIONS: The model simulation suggested that RDTs could significantly reduce P. vivax malaria incidence and medical costs. Moreover, cost-benefit analysis demonstrated that the introduction of RDTs was beneficial over microscopy alone. These results support the need for widespread adoption of RDTs.

Glycogen Synthase Kinase 3ß Regulates Antiviral Responses of TLR3 via TRAF2-Src Axis.

The protein tyrosine kinase Src regulates the synthesis of TLR3-mediated IFN-ß via the TBK1-IFN regulatory factor 3 axis. However, the molecular mechanisms regulating Src activity in TLR3 signaling remain unclear. In this study, we report that GSK3ß regulates Src phosphorylation via TNFR-associated factor 2 (TRAF2)-mediated Src ubiquitination. GSK3ß deficiency in mouse embryonic fibroblasts significantly reduces polyinosinic:polycytidylic acid-induced IFN-ß and IFN-stimulated gene expression, which is caused by diminished phosphorylation of Src at tyrosine 416. Src undergoes polyinosinic:polycytidylic acid-dependent lysine 63 chain ubiquitination, and TRAF2 is a direct E3 ligase for Src. Our study reveals novel mechanisms underlying TLR3-mediated antiviral responses mediated via the GSK3ß-TRAF2-Src axis.

RACK1 interaction with c-Src is essential for osteoclast function.

The scaffolding protein receptor for activated C-kinase 1 (RACK1) mediates receptor activator of nuclear factor κΒ ligand (RANKL)-dependent activation of p38 MAPK in osteoclast precursors; however, the role of RACK1 in mature osteoclasts is unclear. The aim of our study was to identify the interaction between RACK1 and c-Src that is critical for osteoclast function. A RACK1 mutant protein (mutations of tyrosine 228 and 246 residues to phenylalanine; RACK1 Y228F/Y246F) did not interact with c-Src. The mutant retained its ability to differentiate into osteoclasts; however, the integrity of the RANKL-mediated cytoskeleton, bone resorption activity, and phosphorylation of c-Src was significantly decreased. Importantly, lysine 152 (K152) within the Src homology 2 (SH2) domain of c-Src is involved in RACK1 binding. The c-Src K152R mutant (mutation of lysine 152 into arginine) impaired the resorption of bone by osteoclasts. These findings not only clarify the role of the RACK1-c-Src axis as a key regulator of osteoclast function but will also help to develop new antiresorption therapies to prevent bone loss-related diseases.

Reciprocal regulation of TLR2-mediated IFN-ß production by SHP2 and Gsk3ß.

Toll-like receptor 2 (TLR2) mediates the innate immune response to bacterial lipopeptides and peptidoglycans by stimulating the production of inflammatory cytokines. However, the mechanisms by which TLR2 signaling regulates type I interferon (IFN)-ß production are poorly understood. Here, we identified Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) as a negative regulator of TLR2-induced IFN-ß production. Pharmacological inhibition or reduced expression of SHP2 potentiated TLR2 agonist-mediated IFN-ß transcription and STAT1 activation, whereas overexpression of SHP2 impaired IFN-ß transcription and STAT1 activation. SHP2 physically associated with the glycogen synthase kinase 3ß (Gsk3ß) in an agonist-dependent manner. Gsk3ß positively regulates transcription of IFN-ß following TLR2 stimulation by inhibiting the phosphorylation of SHP2. SHP2 inhibited the transcriptional activity of IRF-1 and IRF-8 at the IFN-ß promoter. Remarkably, IRF-1 and IRF-8 are recruited to the IFN-ß promoter in a SHP2 phosphatase activity-dependent manner. These findings provide insight into the mechanisms by which SHP2 and Gsk3ß work together to modulate TLR2-mediated IFN-ß production in macrophages.

FlexPro MD, a Mixture of Krill Oil, Astaxanthin, and Hyaluronic Acid, Suppresses Lipopolysaccharide-Induced Inflammatory Cytokine Production Through Inhibition of NF-κB.

FlexPro MD® (FP-MD), a novel multi-ingredient dietary supplement formulation, has been demonstrated to relieve knee joint pain in humans. However, the mechanisms of action responsible for the activity of FP-MD have not been elucidated. In this study, we show the anti-inflammatory effects of FP-MD in RAW264.7 macrophage cells and mice challenged with lipopolysaccharide (LPS). FP-MD significantly inhibited the mRNA levels of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1ß. In contrast, it elevated the mRNA levels of anti-inflammatory cytokine IL-10 in LPS-stimulated RAW264.7 cells. FP-MD markedly reduced LPS-induced phosphorylation levels of nuclear factor-κB (NF-κB) p65 and inhibitor of κB-α (IκB-α). Importantly, the anti-inflammatory effects of FP-MD were demonstrated in mice with LPS-induced inflammatory arthritis in which FP-MD significantly reduced the expression levels of pro-inflammatory cytokines and inflammatory markers. Thus, this study suggests that FP-MD has anti-inflammatory effects by inhibiting NF-κB that may offer a molecular basis for its pain relief property.

Sirt6 cooperates with Blimp1 to positively regulate osteoclast differentiation.

Global deletion of the gene encoding a nuclear histone deacetylase sirtuin 6 (Sirt6) in mice leads to osteopenia with a low bone turnover due to impaired bone formation. But whether Sirt6 regulates osteoclast differentiation is less clear. Here we show that Sirt6 functions as a transcriptional regulator to directly repress anti-osteoclastogenic gene expression. Targeted ablation of Sirt6 in hematopoietic cells including osteoclast precursors resulted in increased bone volume caused by a decreased number of osteoclasts. Overexpression of Sirt6 led to an increase in osteoclast formation, and Sirt6-deficient osteoclast precursor cells did not undergo osteoclast differentiation efficiently. Moreover, we showed that Sirt6, induced by RANKL-dependent NFATc1 expression, forms a complex with B lymphocyte-induced maturation protein-1 (Blimp1) to negatively regulate expression of anti-osteoclastogenic gene such as Mafb. These findings identify Sirt6 as a novel regulator of osteoclastogenesis by acting as a transcriptional repressor.

Sirtuin 3 (SIRT3) maintains bone homeostasis by regulating AMPK-PGC-1ß axis in mice.

The mitochondrial sirtuin 3 (SIRT3) is involved in suppressing the onset of multiple pathologies, including cardiovascular disease, fatty liver, age-related hearing loss, and breast cancer. But a physiological role of SIRT3 in bone metabolism is not known. Here we show that SIRT3 is a key regulatory molecule to maintain bone homeostasis. Mice deficient in SIRT3 exhibited severe osteopenia owing to increased numbers of osteoclasts. Osteoclast precursors from Sirt3-/- mice underwent increased osteoclastogenesis in response to receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclast differentiation. SIRT3 expression from RANKL induction depended on the transcription coactivator PGC-1ß (peroxisome proliferator-activated receptor-γ co-activator-1ß) and the nuclear receptor ERRα (estrogen receptor-related receptor α), and that SIRT3 inhibited the differentiation by interfering with the RANKL-induced expression of PGC-1ß. Thus an auto-regulatory feedback mechanism operates to induce its own inhibitor SIRT3 by PGC-1ß. Moreover, Sirt3-/- osteoclast precursors reduced AMP-activated protein kinase (AMPK) phosphorylation through down-regulating the expression of AMPK. Our results suggest that a mitochondrial SIRT3 is an intrinsic inhibitor for RANKL-mediated osteoclastogenesis.

Glycogen synthase kinase 3ß in Toll-like receptor signaling.

Toll-like receptors (TLRs) play a critical role in the innate immune response against pathogens. Each TLR recognizes specific pathogen-associated molecular patterns, after which they activate the adaptor protein MyD88 or TRIF-assembled signaling complex to produce immune mediators, including inflammatory cytokines and type I IFNs. Although the activation of TLR is important for host defense, its uncontrolled activation can damage the host. During the past decade, numerous studies have demonstrated that GSK3ß is a key regulator of inflammatory cytokine production in MyD88-mediated TLR signaling via TLR2 and TLR4. Recently, GSK3ß has also been implicated in the TRIF-dependent signaling pathway via TLR3. In this review, we describe current advances on the regulatory role of GSK3ß in immune responses associated with various TLRs. A better understanding of the role of GSK3ß in TLR signaling might lead to more effective anti-inflammatory interventions. [BMB Reports 2016; 49(6): 305-310].

Glycogen synthase kinase 3ß ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production.

TRAF6 is critical for the production of inflammatory cytokines in various TLR-mediated signalling pathways. However, it is poorly understood how TRAF6 regulates TLR3 responses. Here we demonstrate that GSK3ß interacts with TRAF6 and positively regulates the TLR3-mediated signalling. Suppression of GSK3ß expression or its kinase activity drastically reduces the production of inflammatory cytokines and the induction of c-Fos by decreasing ERK and p38 phosphorylation. GSK3ß physically associates with TRAF6 in a TLR3 ligand poly I:C-dependent manner. TRAF6 is determined to be a direct E3 ligase for GSK3ß, and TRAF6-mediated GSK3ß ubiquitination is essential for poly I:C-dependent cytokine production by promoting the TLR3 adaptor protein TRIF-assembled signalling complex.

Glycogen synthase kinase 3ß promotes osteogenic differentiation of murine adipose-derived stromal cells.

Although the role of glycogen synthase kinase 3ß (GSK3ß) in osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) is well-characterized as a negative regulator of ß-catenin, its effect on osteogenesis of adipose-derived stromal cells (ADSCs) is poorly understood. Here, we show that GSK3ß positively regulates osteogenic differentiation of murine ADSCs. Gain-of-function studies showed that GSK3ß promotes in vitro osteogenesis of ADSCs. Regulation of GSK3ß activity in ADSCs, either by small interfering RNA (siRNA)-mediated GSK3ß silencing or by pharmacological inhibitors, blunted osteogenesis and the expression of osteogenic markers. Importantly, we demonstrated that transgenic mice, engineered to overexpress the constitutively active GSK3ß (GSK3ß-S9A) mutant, exhibited a marked increase in osteogenesis, whereas expression of the catalytically inactive GSK3ß (GSK3ß-K85A) in mice inhibits osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by GSK3ß was mediated by downregulation of ß-catenin. Remarkably, ß-catenin silencing enhances osteogenesis and osteoblast marker gene expression such as alkaline phosphatase (ALP) and osterix. Taken together, these findings demonstrate a novel role for GSK3ß in the regulation of osteogenic differentiation in ADSCs.

Inactivation of glycogen synthase kinase-3ß is required for osteoclast differentiation.

Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3ß in osteoblasts is well characterized as a negative regulator of ß-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3ß inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3ß activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3ß (GSK3ß-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3ß (GSK3ß-K85R) or small interfering RNA (siRNA)-mediated GSK-3ß silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3ß further confirmed the negative role of GSK-3ß in osteoclast formation. We also show that overexpression of the GSK3ß-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3ß-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3ß in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.

GSK3beta Inhibitor Peptide Protects Mice from LPS-induced Endotoxin Shock.

BACKGROUND: Glycogen synthase kinase 3beta (GSK3beta) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological GSK3beta inhibitors in rodent models of septic shock. Since most of the GSK3beta inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive GSK3beta inhibitors is needed. METHODS: Based on the unique phosphorylation motif of GSK3beta, we designed and generated a novel class of GSK3beta inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival. RESULTS: We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock. CONCLUSION: Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.