USP18 induction regulates immunometabolism to attenuate M1 signal-polarized macrophages and enhance IL-4-polarized macrophages in systemic lupus erythematosus.

Effective treatment of systemic lupus erythematosus (SLE) remains an unmet need. Different subsets of macrophages play differential roles in SLE and the modulation of macrophage polarization away from M1 status is beneficial for SLE therapeutics. Given the pathogenic roles of type I interferons (IFN-I) in SLE, this study investigated the effects and mechanisms of a mitochondria localization molecule ubiquitin specific peptidase 18 (USP18) preserving anti-IFN effects and isopeptidase activity on macrophage polarization. After observing USP18 induction in monocytes from SLE patients, we studied mouse bone marrow-derived macrophages and showed that USP18 deficiency increased M1signal (LPS + IFN-γ treatment)-induced macrophage polarization, and the effects involved the induction of glycolysis and mitochondrial respiration and the expression of several glycolysis-associated enzymes and molecules, such as hypoxia-inducible factor-1α. Moreover, the effects on mitochondrial activities, such as mitochondrial DNA release and mitochondrial reactive oxygen species production were observed. In contrast, the overexpression of USP18 inhibited M1signal-mediated and enhanced interleukin-4 (IL-4)-mediated polarization of macrophages and the related cellular events. Moreover, the levels of USP18 mRNA expression showed tendency of correlation with the expression of metabolic enzymes in monocytes from patients with SLE. We thus concluded that by preserving anti-IFN effect and downregulating M1 signaling, promoting USP18 activity may serve as a useful approach for SLE therapeutics.

Epigallocatechin-3-gallate exhibits immunomodulatory effects in human primary T cells.

T cells secrete several inflammatory cytokines that play a critical role in the progression of atherosclerosis. Although green tea epigallocatechin-3-gallate (EGCG) exerts anti-inflammatory and anti-atherosclerotic effects in animals, few studies have identified the mechanism underlying these effects in human primary T cells. This study investigated the pathway involved in EGCG modulation of cytokine secretion in activated human primary T cells. We pre-treated human primary T cells with EGCG (0.1, 1, 5, 10, and 20 µM) for 4 h and incubated them with or without phorbol 12-myristate 13-acetate and ionomycin (P/I) for 20 h. The cytokine production, activator protein (AP)-1 binding activity, and level of mitogen-activated protein kinase (MAPK) were assessed using enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blotting, respectively. At 10 and 20 µM, EGCG decreased interleukin (IL)-2 levels by 26.0% and 38.8%, IL-4 levels by 41.5% and 55.9%, INF-γ levels by 31.3% and 34.7%, and tumor-necrosis factor (TNF)-α levels by 23.0% and 37.6%, respectively. In addition, the level of phosphorylated c-Jun N-terminal (p-JNK) and extracellular signal-regulated kinase (p-ERK) was decreased, but not the level of p-p38 MAPK. EGCG did not alter any of the total protein amounts, suggesting a selective effect on specific types of MAPKs in stimulated human T cells. EGCG tended to inactivate AP-1 DNA-binding activity. The P/I-induced production of IL-2, IL-4, INF-γ, and TNF-α by human T cells was suppressed by AP-1 inhibitor in a concentration-dependent manner. In conclusion, EGCG suppressed cytokine secretion in activated human primary T cells, and this effect was likely mediated by AP-1 inactivation through the ERK and JNK, but not p38 MAPK, pathways. These results may be related to the mechanisms through which EGCG inhibits immune- or inflammation-related atherogenesis.

Compound K inhibits priming and mitochondria-associated activating signals of NLRP3 inflammasome in renal tubulointerstitial lesions.

BACKGROUND: Renal tubulointerstitial lesions (TILs), a key pathological hallmark for chronic kidney disease to progress to end-stage renal disease, feature renal tubular atrophy, interstitial mononuclear leukocyte infiltration and fibrosis in the kidney. Our study tested the renoprotective and therapeutic effects of compound K (CK), as described in our US patent (US7932057B2), on renal TILs using a mouse unilateral ureteral obstruction (UUO) model. METHODS: Renal pathology was performed and renal draining lymph nodes were subjected to flow cytometry analysis. Mechanism-based experiments included the analysis of mitochondrial dysfunction, a model of tubular epithelial cells (TECs) under mechanically induced constant pressure (MICP) and tandem mass tags (TMT)-based proteomics analysis. RESULTS: Administration of CK ameliorated renal TILs by reducing urine levels of proinflammatory cytokines, and preventing mononuclear leukocyte infiltration and fibrosis in the kidney. The beneficial effects clearly correlated with its inhibition of: (i) NF-κB-associated priming and the mitochondria-associated activating signals of the NLRP3 inflammasome; (ii) STAT3 signalling, which in part prevents NLRP3 inflammasome activation; and (iii) the TGF-ß-dependent Smad2/Smad3 fibrotic pathway, in renal tissues, renal TECs under MICP and/or activated macrophages, the latter as a major inflammatory player contributing to renal TILs. Meanwhile, TMT-based proteomics analysis revealed downregulated renal NLRP3 inflammasome activation-associated signalling pathways in CK-treated UUO mice. CONCLUSIONS: The present study, for the first time, presents the potent renoprotective and therapeutic effects of CK on renal TILs by targeting the NLRP3 inflammasome and STAT3 signalling.

Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells.

Toll-like receptors (TLRs) are important sensors that recognize pathogen-associated molecular patterns. Generally, TLR9 is known to recognize bacterial or viral DNA but not viral RNA and initiate an immune response. Herein, we demonstrate that infection with dengue virus (DENV), an RNA virus, activates TLR9 in human dendritic cells (DCs). DENV infection induces release of mitochondrial DNA (mtDNA) into the cytosol and activates TLR9 signaling pathways, leading to production of interferons (IFNs). The DENV-induced mtDNA release involves reactive oxygen species generation and inflammasome activation. DENV infection disrupts the association between transcription factor A mitochondria (TFAM) and mtDNA and activates the mitochondrial permeability transition pores. The side-by-side comparison of TLR9 and cyclic GMP-AMP synthase (cGAS) knockdown reveals that both cGAS and TLR9 comparably contribute to DENV-induced immune activation. The significance of TLR9 in DENV-induced immune response is also confirmed in examination with the bone marrow-derived DCs prepared from Tlr9-knockout mice. Our study unravels a previously unrecognized phenomenon in which infection with an RNA virus, DENV, activates TLR9 signaling by inducing mtDNA release in human DCs.

Anti-inflammatory and anti-osteoarthritis effects of Cm-02 and Ck-02.

Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive deterioration of articular cartilage. There have been reports that small molecule inhibitors have anti-osteoarthritis effects; however, the effects of 3-(4-chloro-2-fluorophenyl)-6-(2,4-difluorophenyl)-2H-benzo[e] [1,3]oxazine-2,4(3H)-dione (Cm-02) and 6-(2,4-difluorophenyl)-3-(3,4-difluorophenyl)-2H-benzo[e] [1,3]oxazine-2,4(3H)-dione (Ck-02), small molecule inhibitors which share many structural similarities with quercetin (a potent anti-inflammatory flavonoid), remain unclear. In this study, TNF-α-stimulated porcine and human chondrocyte models were used to investigate the inhibitory effects of Cm-02 and Ck-02 on the molecular mechanisms underlying the anti-OA effects. TNF-α was used to stimulate porcine and human chondrocytes to mimic immunomodulatory potency in-vitro. Anti-osteoarthritic effects were characterized in terms of protein and mRNA levels associated with the pathogenesis of OA. We also examined (1) the inducible nitric oxide synthase (iNOS)-nitric oxide (NO) system in cultured chondrocytes, (2) matrix metalloproteinases (MMPs) in cultured chondrocytes, and (3) aggrecan degradation in cartilage explants. Finally, we tested the activation of nuclear factor-kappaB (NF-κB), interferon regulatory factor-1 (IRF-1), and activate the protein-1 (AP-1), and we tested the signal transduction and activation of transcription-3 (STAT-3). Our results indicate that, in chondrocytes, Cm-02 and Ck-02 inhibit TNF-α induced NO production, iNOS, MMP, the expression of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and the enzyme activity of MMP-13. Furthermore, both Cm-02 and Ck-02 were found to stimulate TNF-α, which has been shown to suppress the activation of several transcription factors, including NF-κB, STAT-3, and IRF-1 in porcine and human chondrocytes. Cm-02 and Ck-02 were also found to help prevent the release of proteoglycans from cartilage explants. Our findings demonstrate that both Cm-02 and Ck-02 have potent anti-inflammatory activities and the ability to protect cartilage in an OA cell model. These findings indicate that Cm-02 and Ck-02 have the potential to be further developed for the therapeutic treatment of OA.

Pitavastatin Exerts Potent Anti-Inflammatory and Immunomodulatory Effects via the Suppression of AP-1 Signal Transduction in Human T Cells.

Statins inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase are the standard treatment for hypercholesterolemia in atherosclerotic cardiovascular disease (ASCVD), mediated by inflammatory reactions within vessel walls. Several studies highlighted the pleiotropic effects of statins beyond their lipid-lowering properties. However, few studies investigated the effects of statins on T cell activation. This study evaluated the immunomodulatory capacities of three common statins, pitavastatin, atorvastatin, and rosuvastatin, in activated human T cells. The enzyme-linked immunosorbent assay (ELISA) and quantitative real time polymerase chain reaction (qRT-PCR) results demonstrated stronger inhibitory effects of pitavastatin on the cytokine production of T cells activated by phorbol 12-myristate 13-acetate (PMA) plus ionomycin, including interleukin (IL)-2, interferon (IFN)-γ, IL-6, and tumor necrosis factor α (TNF-α). Molecular investigations revealed that pitavastatin reduced both activating protein-1 (AP-1) DNA binding and transcriptional activities. Further exploration showed the selectively inhibitory effect of pitavastatin on the signaling pathways of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK). Our findings suggested that pitavastatin might provide additional benefits for treating hypercholesterolemia and ASCVD through its potent immunomodulatory effects on the suppression of ERK/p38/AP-1 signaling in human T cells.

Chondroprotective Effects and Mechanisms of Dextromethorphan: Repurposing Antitussive Medication for Osteoarthritis Treatment.

Osteoarthritis (OA) is the most common joint disorder and primarily affects older people. The ideal anti-OA drug should have a modest anti-inflammatory effect and only limited or no toxicity for long-term use. Because the antitussive medication dextromethorphan (DXM) is protective in atherosclerosis and neurological diseases, two common disorders in aged people, we examined whether DXM can be protective in pro-inflammatory cytokine-stimulated chondrocytes and in a collagen-induced arthritis (CIA) animal model in this study. Chondrocytes were prepared from cartilage specimens taken from pigs or OA patients. Western blotting, quantitative PCR, and immunohistochemistry were adopted to measure the expression of collagen II (Col II) and matrix metalloproteinases (MMP). DXM significantly restored tumor necrosis factor-alpha (TNF-α)-mediated reduction of collagen II and decreased TNF-α-induced MMP-13 production. To inhibit the synthesis of MMP-13, DXM blocked TNF-α downstream signaling, including I kappa B kinase (IKK)α/ß-IκBα-nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)-activator protein-1 (AP-1) activation. Besides this, DXM protected the CIA mice from severe inflammation and cartilage destruction. DXM seemed to protect cartilage from inflammation-mediated matrix degradation, which is an irreversible status in the disease progression of osteoarthritis. The results suggested that testing DXM as an osteoarthritis therapeutic should be a focus in further research.

Arthroprotective Effects of Cf-02 Sharing Structural Similarity with Quercetin.

In this study, we synthesized hundreds of analogues based on the structure of small-molecule inhibitors (SMIs) that were previously identified in our laboratory with the aim of identifying potent yet safe compounds for arthritis therapeutics. One of the analogues was shown to share structural similarity with quercetin, a potent anti-inflammatory flavonoid present in many different fruits and vegetables. We investigated the immunomodulatory effects of this compound, namely 6-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione (Cf-02), in a side-by-side comparison with quercetin. Chondrocytes were isolated from pig joints or the joints of patients with osteoarthritis that had undergone total knee replacement surgery. Several measures were used to assess the immunomodulatory potency of these compounds in tumor necrosis factor (TNF-α)-stimulated chondrocytes. Characterization included the protein and mRNA levels of molecules associated with arthritis pathogenesis as well as the inducible nitric oxide synthase (iNOS)⁻nitric oxide (NO) system and matrix metalloproteinases (MMPs) in cultured chondrocytes and proteoglycan, and aggrecan degradation in cartilage explants. We also examined the activation of several important transcription factors, including nuclear factor-kappaB (NF-κB), interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription-3 (STAT-3), and activator protein-1 (AP-1). Our overall results indicate that the immunomodulatory potency of Cf-02 is fifty-fold more efficient than that of quercetin without any indication of cytotoxicity. When tested in vivo using the induced edema method, Cf-02 was shown to suppress inflammation and cartilage damage. The proposed method shows considerable promise for the identification of candidate disease-modifying immunomodulatory drugs and leads compounds for arthritis therapeutics.

JNK/AP-1 activation contributes to tetrandrine resistance in T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a challenging malignancy with a high relapse rate attributed to drug resistance. Tetrandrine (TET), a bisbenzylisoquinoline alkaloid extracted from a Chinese herb, is a potential anti-cancer and anti-leukaemic drug. In this study we investigated the mechanisms of TET resistance in T-ALL cells in vitro. Among the four T-ALL cell lines tested, Jurkat and CEM cells exhibited the lowest and highest resistance to TET with IC50 values at 24 h of 4.31±0.12 and 16.53±3.32 µmol/L, respectively. When treated with TET, the activity of transcription factor activator protein 1 (AP-1) was significantly decreased in Jurkat cells but nearly constant in CEM cells. To avoid cell-specific variation in drug resistance and transcription factor activities, we established a TET-R Jurkat subclone with the estimated IC50 value of 10.90±.92 µmol/L by exposing the cells to increasing concentrations of TET. Interestingly, when treated with TET, TET-R Jurkat cells exhibited enhanced AP-1 and NF-κB activity, along with upregulation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways, whereas the expression of P-gp was not altered. Selective inhibition of JNK but not ERK suppressed AP-1 activity and TET resistance in TET-R Jurkat cells and in CEM cells. These results demonstrate that Jurkat cells acquire TET resistance through activation of the JNK/AP-1 pathway but not through P-gp expression. The JNK/AP-1 pathway may be a potential therapeutic target in relapsed T-ALL.

Translational Implication of Galectin-9 in the Pathogenesis and Treatment of Viral Infection.

The interaction between galectin-9 and its receptor, Tim-3, triggers a series of signaling events that regulate immune responses. The expression of galectin-9 has been shown to be increased in a variety of target cells of many different viruses, such as hepatitis C virus (HCV), hepatitis B virus (HBV), herpes simplex virus (HSV), influenza virus, dengue virus (DENV), and human immunodeficiency virus (HIV). This enhanced expression of galectin-9 following viral infection promotes significant changes in the behaviors of the virus-infected cells, and the resulting events tightly correlate with the immunopathogenesis of the viral disease. Because the human immune response to different viral infections can vary, and the lack of appropriate treatment can have potentially fatal consequences, understanding the implications of galectin-9 is crucial for developing better methods for monitoring and treating viral infections. This review seeks to address how we can apply the current understanding of galectin-9 function to better understand the pathogenesis of viral infection and better treat viral diseases.

Gentiana scabra Reduces SR-A Expression and Oxidized-LDL Uptake in Human Macrophages.

BACKGROUND: Macrophages can imbibe low-density lipoprotein (LDL) through scavenger receptors to become foam cells, which is critical in the initiation and progression of atherosclerosis. Mounting evidence suggests that the anti-inflammatory nature of Chinese herbs have the capacity to halt the complex mechanisms underlying atherosclerosis. This study examined the effects of Chinese herbs on foam cell formation. METHODS: Chinese herbs were obtained from the Sun Ten pharmaceutic company. Using oxidized LDL (OxLDL) uptake and a cell toxicity assay, we screened more than 30 types of Chinese herbs. Western blotting was used to determine expressions of scavenger receptors (SRs) and extracellular-signal-regulated kinase (ERK) activities. RESULTS: We found that Gentiana scabra reduced oxidized LDL uptake effectively in THP-1 macrophages (p < 0.05 vs. OxLDL treated control). Moreover, treatment with Gentiana scabra in THP-1 macrophages resulted in decreased expression of scavenger receptor- A (SR-A) (p < 0.05 vs. control). Molecular investigation revealed that Gentiana scabra inhibited SR-A protein expression, possibly by regulating ERK signaling pathways (p < 0.05 vs. control). CONCLUSIONS: By regulating SR-A expression, Gentiana scabra reduced oxidized LDL uptake in human macrophages. These results support the potential use of Gentiana scabra in treating atherosclerosis.

Up-regulation of galectin-9 induces cell migration in human dendritic cells infected with dengue virus.

Galectin-9 (Gal-9) exerts immunosuppressive effects by inducing apoptosis in T cells that produce interferon-γ and interleukin (IL)-17. However, Gal-9 can be pro-inflammatory in lipopolysaccharide-stimulated monocytes. Using microarray analysis, we observed that Gal-9 was up-regulated in human dendritic cells (DCs) after dengue virus (DV) infection. The investigation into the immunomodulatory effects and mechanisms of Gal-9 in DCs exposed to DV revealed that DV infection specifically increased mRNA and protein levels of Gal-9 but not those of Gal-1 or Gal-3. Blocking p38, but not c-Jun N-terminal kinase or extracellular signal-regulated kinase (ERK), inhibited DV-induced expression of Gal-9. Reduction in Gal-9 by small interference RNA treatment suppressed DV-stimulated migration of DCs towards the chemoattractants CCL19 and CCL21. In addition, DV-induced IL-12p40 production was reduced after knockdown of Gal-9 in DCs. Furthermore, Gal-9 deficiency suppressed DV-induced activation of nuclear factor-κB. Inhibition of DV-induced DC migration under conditions of Gal-9 deficiency was mediated through suppressing ERK activation but not by regulating the expression of CCR7, the receptor for CCL19 and CCL21. Both the reduction in IL-12 production and the suppression of ERK activity might account for the inhibition of DV-induced DC migration after knockdown of Gal-9. In summary, this study reveals the roles of Gal-9 in DV-induced migration of DCs. The findings indicate that Gal-9 might be a therapeutic target for preventing immunopathogenesis induced by DV infection.

Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory "Junn-Chenn-Zuou-SS".

BACKGROUND: For thousands of years, it remains unclear why Chinese prefer complex herbal remedy and seldom try to purify it. One of the reasons is that they believe Chinese herbs compared to Western drugs are relatively less toxic and better tolerated. The so called "Junn-Chenn-Zuou-SS" theory illustrates a concept of coordinated effects from a combination of different Chinese herbs. PG27, a refined extract from a well-known Chinese antirheumatic herb Tripterygium wilfordii Hook f (TwHf), is effective in attenuating transplantation rejection and extending survival of cardiac xenografts. METHODS: Experiments were conducted in human primary T lymphocytes isolated from buffy coat. The activities of the inhibitor of kappaB alpha kinase-inhibitor of kappaB alpha-nuclear factor kappaB (IKK-IκBα-NF-κB) and mitogen activated protein kinase-activator protein-1 (MAPK-AP-1) signaling pathways were determined via electrophoretic mobility shift assays, immunoprecipitation kinase assays, Western blots, and transfection assays. RESULTS: We showed that PG27 inhibited IKKα-IκBα-NF-κB and MAPK-AP-1 signaling pathways; however, IKKß activity was less susceptible to inhibition by PG27. In contrast, the purified component of TwHf, PG490 (triptolide), reduced both MAPK-AP-1 and IKK-IκBα-NF-κB signaling pathways, including both IKKα and IKKß, with similar potency. By means of high performance liquid chromatography analysis, it was estimated that PG490 constituted 1.27 ± 0.06% of the total PG27 content. Further analysis demonstrated that compared to PG490 alone, PG27 that contained an equal amount of PG490 was less toxic and less immunosuppressive, suggesting the presence of cytoprotective ingredient(s) in the non-PG490 components of PG27. CONCLUSIONS: In addition to demonstrating the immunomodulatory capacity of PG27 as the potential therapeutics for arthritis and prevention of transplantation rejection, the differential regulatory effects and mechanisms by PG27 and PG490 further support in part a possibly-existing Chinese herbal theory "Junn-Chenn-Zuou-SS".

USP18 enhances dengue virus replication by regulating mitochondrial DNA release.

Dengue virus (DENV) infection remains a challenging health threat worldwide. Ubiquitin-specific protease 18 (USP18), which preserves the anti-interferon (IFN) effect, is an ideal target through which DENV mediates its own immune evasion. However, much of the function and mechanism of USP18 in regulating DENV replication remains incompletely understood. In addition, whether USP18 regulates DENV replication merely by causing IFN hyporesponsiveness is not clear. In the present study, by using several different approaches to block IFN signaling, including IFN neutralizing antibodies (Abs), anti-IFN receptor Abs, Janus kinase inhibitors and IFN alpha and beta receptor subunit 1 (IFNAR1)knockout cells, we showed that USP18 may regulate DENV replication in IFN-associated and IFN-unassociated manners. Localized in mitochondria, USP18 regulated the release of mitochondrial DNA (mtDNA) to the cytosol to affect viral replication, and mechanisms such as mitochondrial reactive oxygen species (mtROS) production, changes in mitochondrial membrane potential, mobilization of calcium into mitochondria, 8-oxoguanine DNA glycosylase 1 (OGG1) expression, oxidation and fragmentation of mtDNA, and opening of the mitochondrial permeability transition pore (mPTP) were involved in USP18-regulated mtDNA release to the cytosol. We therefore identify mitochondrial machineries that are regulated by USP18 to affect DENV replication and its association with IFN effects.

Immunosuppressive effects and mechanisms of leflunomide in dengue virus infection of human dendritic cells.

BACKGROUND: Dengue virus (DENV) infection is a serious public health issue without specific treatment. We examined the potential immunomodulatory effects of leflunomide, a dihydroorotate dehydrogenase inhibitor commonly prescribed for arthritis, in DENV-stimulated monocyte-derived dendritic cells (mo-DCs). METHODS: mo-DCs were prepared from purified monocytes. Cytokine and chemokine concentrations were determined by enzyme-linked immunosorbent assay. Expression of cell surface markers or viral E protein was measured by flow cytometry. The activation of transcription factors and kinases was determined by electrophoretic mobility shift assays, Western blotting, or immunoprecipitation kinase assays. Chemotaxis assays were used to determine cell migration. RESULTS: Leflunomide at therapeutic concentrations inhibited cytokine and chemokine production from DENV-infected mo-DCs. Leflunomide suppressed mo-DC maturation by downregulating the expression of both CD80 and CD86. In addition, leflunomide inhibited DENV-induced mo-DC migration and mo-DC response to chemoattractants CCL19 and CCL21. Inhibition of mo-DC migration was likely due to the suppression of CCR7 expression on mo-DCs. These events were associated with the suppression of nuclear factor kappa B and activator protein-1 signaling pathways by leflunomide. CONCLUSIONS: Leflunomide preserves immunosuppressive effects, inhibiting activation of DENV-stimulated mo-DCs. Leflunomide may be helpful in the development of therapeutics for DENV infection.

A benzamide-linked small molecule HS-Cf inhibits TNF-α-induced interferon regulatory factor-1 in porcine chondrocytes: a potential disease-modifying drug for osteoarthritis therapeutics.

BACKGROUND: Using tumor necrosis factor-alpha (TNF-α)-activated porcine chondrocytes as a screening tool, we aim to synthesize and identify small-molecule inhibitors preserving immunomodulatory effects as therapeutics for osteoarthritis (OA). METHODS: Chondrocytes were isolated from pig joints. A minilibrary of 300 benzamide-linked small molecules was established. The levels of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) were measured by Western blot and Griess reaction, respectively. Proteoglycan degradation in cartilage explants was determined by histochemistry analysis. The activation of transcription factors and protein kinases was determined by electrophoretic mobility shift assays or Western blots. Zymography and real-time reverse transcriptase-polymerase chain reaction were used to determine enzyme activity and expression of matrix metalloproteinases (MMPs) and aggrecanases, respectively. RESULTS: Bioassay screening of benzamide-linked small molecules revealed that 2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide (HS-Cf) was a potent inhibitor of NO production and iNOS expression in TNF-α-stimulated porcine chondrocytes. HS-Cf suppressed TNF-α-induced activity of MMP-13 and expressions of several aggrecanases and prevented TNF-α-mediated reduction of collagen II. Histochemistry analysis confirmed that HS-Cf could prevent TNF-α-induced degradation and release of proteoglycan/aggrecan in cartilage explants. Such effects by HS-Cf were likely through suppressing TNF-α-induced interferon regulatory factor-1 (IRF-1) but not nuclear factor-kappaB signaling. The significance of IRF-1 was further confirmed by short hairpin knockdown studies. CONCLUSIONS: In a minilibrary containing 300 small molecules, we identified a benzamide-linked small molecule, HS-Cf, that through down-regulating TNF-α-induced IRF-1 activity suppressed chondrocyte activation and prevented cartilage destruction. HS-Cf might be a potential disease-modifying drug for OA therapeutics.

Advanced glycation end products cause collagen II reduction by activating Janus kinase/signal transducer and activator of transcription 3 pathway in porcine chondrocytes.

OBJECTIVES: The major risk factor for OA is ageing; however, the mechanisms remain largely unclear. We investigated the effects and mechanisms of advanced glycation end products (AGEs) that accumulate in aged joints in chondrocytes. METHODS: Porcine chondrocytes or cartilage fragments were prepared. Gene expression of MMPs and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) was assessed by real-time RT-PCR. Gelatin zymography was used to determine MMP-13 enzyme activity. Histochemistry or immunoblotting analysis was applied to determine the expression of collagen II, proteoglycan and aggrecan. Electrophoretic mobility shift assay and immunoblotting were used to study the activation of signal transducer and activator of transcription 3 (STAT3). Genetic manipulations with short hairpin RNA (shRNA) or dominant negative constructs were applied. RESULTS: AGE enhanced expression and enzyme activity of MMP and ADAMTS genes and resulted in reduction of collagen II. Both janus kinase 2 (JAK2) and JAK3 inhibitors suppressed AGE-induced MMP-13, ADAMTS-4 and ADAMTS-5 expression and enzyme activity. Inhibition of JAK2 or JAK3 prevented AGE-mediated decrease of collagen II in chondrocytes and proteoglycan (aggrecan) degradation in cartilage fragments. In addition, interference of STAT3 expression inhibited AGE-induced MMP-13 and ADAMTS enzyme activities and mRNA levels. Furthermore, expression of the dominant negative receptor of AGE (DN-RAGE) blocked AGE-induced STAT3 phosphorylation. CONCLUSION: Blocking JAK/STAT3 signalling pathway inhibited AGE-induced activation of MMP-13 and ADAMTS and prevented AGE-mediated decrease of collagen II and proteoglycan (aggrecan). The results indicated that JAK/STAT3 pathway may be a potential target for designing disease-modifying drugs for the treatment of OA.

Mitochondrial protein CMPK2 regulates IFN alpha-enhanced foam cell formation, potentially contributing to premature atherosclerosis in SLE.

BACKGROUND: Premature atherosclerosis occurs in patients with SLE; however, the mechanisms remain unclear. Both mitochondrial machinery and proinflammatory cytokine interferon alpha (IFN-α) potentially contribute to atherogenic processes in SLE. Here, we explore the roles of the mitochondrial protein cytidine/uridine monophosphate kinase 2 (CMPK2) in IFN-α-mediated pro-atherogenic events. METHODS: Foam cell measurements were performed by oil red O staining, Dil-oxLDL uptake and the BODIPY approach. The mRNA and protein levels were measured by qPCR and Western blotting, respectively. Isolation of CD4+ T cells and monocytes was performed with monoclonal antibodies conjugated with microbeads. Manipulation of protein expression was conducted by either small interference RNA (siRNA) knockdown or CRISPR/Cas9 knockout. The expression of mitochondrial reactive oxygen species (mtROS) was determined by flow cytometry and confocal microscopy. RESULTS: IFN-α enhanced oxLDL-induced foam cell formation and Dil-oxLDL uptake by macrophages. In addition to IFN-α, several triggers of atherosclerosis, including thrombin and IFN-γ, can induce CMPK2 expression, which was elevated in CD4+ T cells and CD14+ monocytes isolated from SLE patients compared to those isolated from controls. The analysis of cellular subfractions revealed that CMPK2 was present in both mitochondrial and cytosolic fractions. IFN-α-induced CMPK2 expression was inhibited by Janus kinase (JAK)1/2 and tyrosine kinase 2 (Tyk2) inhibitors. Both the knockdown and knockout of CMPK2 attenuated IFN-α-mediated foam cell formation, which involved the reduction of scavenger receptor class A (SR-A) expression. CMPK2 also regulated IFN-α-enhanced mtROS production and inflammasome activation. CONCLUSIONS: The study suggests that CMPK2 plays contributing roles in the pro-atherogenic effects of IFN-α.

Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways.

Mitochondria regulate the immune response after dengue virus (DENV) infection. Microarray analysis of genes identified the upregulation of mitochondrial cytidine/uridine monophosphate kinase 2 (CMPK2) by DENV infection. We used small interfering RNA-mediated knockdown (KD) and CRISPR-Cas9 knockout (KO) approaches, to investigate the role of CMPK2 in mouse and human cells. The results showed that CMPK2 was critical in DENV-induced antiviral cytokine release and mitochondrial oxidative stress and mitochondrial DNA release to the cytosol. The DENV-induced activation of Toll-like receptor (TLR)-9, inflammasome pathway, and cell migration was suppressed by CMPK2 depletion; however, viral production increased under CMPK2 deficiency. Examining mouse bone marrow-derived dendritic cells from interferon-alpha (IFN-α) receptor-KO mice and signal transducer and activator of transcription 1 (STAT1)-KO mice, we confirmed that CMPK2-mediated antiviral activity occurred in IFN-dependent and IFN-independent manners. In sum, CMPK2 is a critical factor in DENV-induced immune responses to determine innate immunity.

Triggering of DC migration by dengue virus stimulation of COX-2-dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation.

A term "bone-breaking fever" is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX-prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX-2 and the production of prostaglandin E2 (PGE2) in DC, and stimulated the DNA binding of NF-kappaB and the kinase activity of both IkappaBalpha kinase (IKK) alpha and beta. DV infection also activated MAPK and AP-1 signaling. Both IkappaBalpha kinase-NF-kappaB and MAPK-AP-1 were upstream of COX-2 activation. Our investigation into the significance of COX-2-PGE2 pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX-2 or MAPK activity suppresses DV-induced DC migration. Our data also suggest that PGE2 can induce CCR7 expression on DC and that antagonists of the PGE2 receptors EP2 and EP4 suppress DV-induced DC migration. We further show that the increased CCR7 expression was observed in both DV-infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX-2 synthesis in DV-infected DC but also the autocrine action of PGE2 on the migration of DV-infected DC.