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1.
JCI Insight ; 5(2)2020 01 30.
Article in English | MEDLINE | ID: mdl-31996485

ABSTRACT

Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal-regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.


Subject(s)
Cell Death/drug effects , Enzyme Inhibitors/pharmacology , Liver Cirrhosis/metabolism , Liver/injuries , Liver/metabolism , MAP Kinase Kinase Kinase 5/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Administration, Oral , Animals , Apoptosis/drug effects , Apoptosis/physiology , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Enzyme Inhibitors/administration & dosage , Female , Gene Expression Regulation , Hepatic Stellate Cells/metabolism , Inflammasomes/metabolism , Inflammation/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Liver Cirrhosis/pathology , MAP Kinase Kinase Kinase 5/genetics , MAP Kinase Kinase Kinase 5/metabolism , Male , Mice , Mice, Inbred NOD , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Phosphorylation , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Biochem Pharmacol ; 151: 282-290, 2018 05.
Article in English | MEDLINE | ID: mdl-29408488

ABSTRACT

Despite improved therapy, rheumatoid arthritis (RA) remains an unmet medical need. Previous efforts to validate therapeutic targets in the mitogen-activated protein kinase (MAPK) family have had minimal success. Therefore, we evaluated the potential for targeting an upstream MAPK, namely apoptosis signal-regulating kinase 1 (ASK1), as an alternative approach. ASK1 protein and gene expression were observed in RA and osteoarthritis (OA) synovium as determined by immunohistochemistry (IHC) and qPCR, respectively, particularly in the synovial intimal lining. For RA, but not OA synovium, ASK1 correlated with IL-1ß and TNF gene expression. ASK1 was also expressed by cultured fibroblast-like synoviocytes (FLS), with significantly higher levels in RA compared with OA cells. IL-1ß and TNF stimulation significantly increased ASK1 expression in a time-and concentration-dependent manner in cultured FLS. ASK1 promoter activity was significantly increased by IL-1ß and TNF and was dependent on an upstream RelA binding motif. A selective small molecule ASK1 inhibitor reduced RA FLS invasion, migration and proliferation in vitro and decreased arthritis severity in the rat collagen-induced arthritis (CIA) model. In summary, our findings demonstrate that ASK1 modulates signaling pathways relevant to RA in vitro and in vivo. It is induced by inflammatory cytokines through the activation of NF-κB, which could provide some site- and event specificity. Thus, inhibitors of the upstream MAPK ASK1 could be a novel approach to treating inflammatory arthritis.


Subject(s)
Arthritis, Rheumatoid/enzymology , MAP Kinase Kinase Kinase 5/metabolism , Osteoarthritis/enzymology , Animals , Arthritis, Experimental/drug therapy , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/pathology , Cells, Cultured , Cytokines/immunology , Enzyme Inhibitors/therapeutic use , Female , Gene Expression Regulation , Humans , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Kinase Kinase 5/genetics , Molecular Targeted Therapy , Osteoarthritis/drug therapy , Osteoarthritis/pathology , Rats, Inbred Lew , Signal Transduction , Synovial Membrane/drug effects , Synovial Membrane/metabolism , Synovial Membrane/pathology , Synoviocytes/drug effects , Synoviocytes/metabolism , Synoviocytes/pathology
3.
J Hepatol ; 68(5): 912-921, 2018 05.
Article in English | MEDLINE | ID: mdl-29247724

ABSTRACT

BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. METHODS: Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. CONCLUSION: The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. LAY SUMMARY: New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.


Subject(s)
Antiviral Agents/pharmacology , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/immunology , Pteridines/pharmacology , Toll-Like Receptor 7/agonists , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Aggregation/drug effects , Cell Aggregation/immunology , Disease Models, Animal , Gene Expression Profiling , Hepatitis B, Chronic/virology , Humans , Liver/drug effects , Liver/immunology , Liver/pathology , Pan troglodytes
4.
Nat Commun ; 7: 13710, 2016 12 14.
Article in English | MEDLINE | ID: mdl-27966531

ABSTRACT

Interstitial fibrosis plays a key role in the development and progression of heart failure. Here, we show that an enzyme that crosslinks collagen-Lysyl oxidase-like 2 (Loxl2)-is essential for interstitial fibrosis and mechanical dysfunction of pathologically stressed hearts. In mice, cardiac stress activates fibroblasts to express and secrete Loxl2 into the interstitium, triggering fibrosis, systolic and diastolic dysfunction of stressed hearts. Antibody-mediated inhibition or genetic disruption of Loxl2 greatly reduces stress-induced cardiac fibrosis and chamber dilatation, improving systolic and diastolic functions. Loxl2 stimulates cardiac fibroblasts through PI3K/AKT to produce TGF-ß2, promoting fibroblast-to-myofibroblast transformation; Loxl2 also acts downstream of TGF-ß2 to stimulate myofibroblast migration. In diseased human hearts, LOXL2 is upregulated in cardiac interstitium; its levels correlate with collagen crosslinking and cardiac dysfunction. LOXL2 is also elevated in the serum of heart failure (HF) patients, correlating with other HF biomarkers, suggesting a conserved LOXL2-mediated mechanism of human HF.


Subject(s)
Amino Acid Oxidoreductases/physiology , Heart Failure/metabolism , Myocardium/pathology , Amino Acid Oxidoreductases/blood , Amino Acid Oxidoreductases/metabolism , Animals , Fibrosis/metabolism , Humans , Mice, Knockout , Myocardium/metabolism , Stress, Physiological
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