Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
1.
Proc Natl Acad Sci U S A ; 121(7): e2310264121, 2024 Feb 13.
Article in English | MEDLINE | ID: mdl-38319963

ABSTRACT

Epigenetic regulation plays a crucial role in the pathogenesis of autoimmune diseases such as inflammatory arthritis. DNA hypomethylating agents, such as decitabine (DAC), have been shown to dampen inflammation and restore immune homeostasis. In the present study, we demonstrate that DAC elicits potent anti-inflammatory effects and attenuates disease symptoms in several animal models of arthritis. Transcriptomic and epigenomic profiling show that DAC-mediated hypomethylation regulates a wide range of cell types in arthritis, altering the differentiation trajectories of anti-inflammatory macrophage populations, regulatory T cells, and tissue-protective synovial fibroblasts (SFs). Mechanistically, DAC-mediated demethylation of intragenic 5'-Cytosine phosphate Guanine-3' (CpG) islands of the transcription factor Irf8 (interferon regulatory factor 8) induced its re-expression and promoted its repressor activity. As a result, DAC restored joint homeostasis by resetting the transcriptomic signature of negative regulators of inflammation in synovial macrophages (MerTK, Trem2, and Cx3cr1), TREGs (Foxp3), and SFs (Pdpn and Fapα). In conclusion, we found that Irf8 is necessary for the inhibitory effect of DAC in murine arthritis and that direct expression of Irf8 is sufficient to significantly mitigate arthritis.


Subject(s)
Arthritis , Azacitidine , Mice , Animals , Decitabine/pharmacology , Azacitidine/pharmacology , Epigenesis, Genetic , DNA Methylation , Interferon Regulatory Factors/metabolism , Inflammation/genetics , Arthritis/genetics , Anti-Inflammatory Agents , Membrane Glycoproteins/metabolism , Receptors, Immunologic/genetics
2.
Elife ; 112022 08 02.
Article in English | MEDLINE | ID: mdl-35916374

ABSTRACT

Osteoarthritis is the most common joint disease in the world with significant societal consequences but lacks effective disease-modifying interventions. The pathophysiology consists of a prominent inflammatory component that can be targeted to prevent cartilage degradation and structural defects. Intracellular metabolism has emerged as a culprit of the inflammatory response in chondrocytes, with both processes co-regulating each other. The role of glutamine metabolism in chondrocytes, especially in the context of inflammation, lacks a thorough understanding and is the focus of this work. We display that mouse chondrocytes utilize glutamine for energy production and anabolic processes. Furthermore, we show that glutamine deprivation itself causes metabolic reprogramming and decreases the inflammatory response of chondrocytes through inhibition of NF-κB activity. Finally, we display that glutamine deprivation promotes autophagy and that ammonia is an inhibitor of autophagy. Overall, we identify a relationship between glutamine metabolism and inflammatory signaling and display the need for increased study of chondrocyte metabolic systems.


Subject(s)
Chondrocytes , Osteoarthritis , Animals , Cartilage , Chondrocytes/metabolism , Glutamine/metabolism , Mice , NF-kappa B/metabolism , Osteoarthritis/metabolism
3.
Bone Res ; 10(1): 12, 2022 Feb 11.
Article in English | MEDLINE | ID: mdl-35145063

ABSTRACT

Osteoarthritis is a joint disease characterized by a poorly-defined inflammatory response that does not encompass a massive immune cell infiltration yet contributes to cartilage degradation and loss of joint mobility, suggesting a chondrocyte intrinsic inflammatory response. Using primary chondrocytes from joints of osteoarthritic mice and patients, we first show that these cells express ample pro-inflammatory markers and RANKL in an NF-κB dependent manner. The inflammatory phenotype of chondrocytes was recapitulated by exposure of chondrocytes to IL-1ß and bone particles, which were used to model bone matrix breakdown products revealed to be present in synovial fluid of OA patients, albeit their role was not defined. We further show that bone particles and IL-1ß can promote senescent and apoptotic changes in primary chondrocytes due to oxidative stress from various cellular sources such as the mitochondria. Finally, we provide evidence that inflammation, oxidative stress and senescence converge upon IκB-ζ, the principal mediator downstream of NF-κB, which regulates expression of RANKL, inflammatory, catabolic, and SASP genes. Overall, this work highlights the capacity and mechanisms by which inflammatory cues, primarily joint degradation products, i.e., bone matrix particles in concert with IL-1ß in the joint microenvironment, program chondrocytes into an "inflammatory phenotype" which inflects local tissue damage.

4.
Methods Mol Biol ; 2366: 267-282, 2021.
Article in English | MEDLINE | ID: mdl-34236644

ABSTRACT

The skeletal system is constantly undergoing turnover in order to create strong, organized structures, requiring the bone breakdown and building properties by osteoclasts and osteoblasts, respectively. However, in pathological disease states, excessive osteoclast activity can cause bone loss leading to increase in morbidity and mortality. Osteoclasts differentiate from macrophages in the presence of various factors. M-CSF is a cytokine that is required to maintain the survival of macrophages. However, RANKL is the critical factor required for differentiation of osteoclasts. RANKL is produced from a variety of different cell types such as osteoblasts and osteocytes. RANKL binds to RANK, its receptor, on the surface of osteoclast precursors, which activates various signaling pathways to drive the transcription and production of genes important for osteoclast formation. The major signaling pathway activated by RANKL-RANK interaction is the NF-κB pathway. The NF-κB pathway is the principle inflammatory response pathway activated by a variety of stimuli such as inflammatory cytokines, genotoxic stress, and other factors. This likely explains the finding that inflammatory diseases often present with some component of increased osteoclast formation and activity, driving bone loss. Determining the signaling mechanisms downstream of RANKL can provide valuable therapeutic targets for the treatment of bone loss in various disease states.


Subject(s)
Signal Transduction , Cell Differentiation , NF-kappa B/metabolism , Osteoclasts/metabolism , Osteogenesis , RANK Ligand/metabolism , TNF Receptor-Associated Factor 6
5.
Nat Commun ; 11(1): 3427, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32647171

ABSTRACT

The contribution of inflammation to the chronic joint disease osteoarthritis (OA) is unclear, and this lack of clarity is detrimental to efforts to identify therapeutic targets. Here we show that chondrocytes under inflammatory conditions undergo a metabolic shift that is regulated by NF-κB activation, leading to reprogramming of cell metabolism towards glycolysis and lactate dehydrogenase A (LDHA). Inflammation and metabolism can reciprocally modulate each other to regulate cartilage degradation. LDHA binds to NADH and promotes reactive oxygen species (ROS) to induce catabolic changes through stabilization of IκB-ζ, a critical pro-inflammatory mediator in chondrocytes. IκB-ζ is regulated bi-modally at the stages of transcription and protein degradation. Overall, this work highlights the function of NF-κB activity in the OA joint as well as a ROS promoting function for LDHA and identifies LDHA as a potential therapeutic target for OA treatment.


Subject(s)
Chondrocytes/metabolism , Lactate Dehydrogenase 5/metabolism , Molecular Targeted Therapy , Osteoarthritis/drug therapy , Reactive Oxygen Species/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Aerobiosis , Animals , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/pathology , Cytoprotection/drug effects , Gene Deletion , Gene Expression Regulation/drug effects , Glycolysis/drug effects , Humans , Inflammation/metabolism , Inflammation/pathology , Interleukin-1beta/pharmacology , Knee Joint/pathology , Menisci, Tibial/surgery , Metabolic Networks and Pathways/drug effects , Mice, Inbred C57BL , NAD/metabolism , NF-kappa B/metabolism , Osteoarthritis/genetics , Osteoarthritis/pathology
6.
Elife ; 92020 03 23.
Article in English | MEDLINE | ID: mdl-32202502

ABSTRACT

Inflammatory osteolysis is governed by exacerbated osteoclastogenesis. Ample evidence points to central role of NF-κB in such pathologic responses, yet the precise mechanisms underpinning specificity of these responses remain unclear. We propose that motifs of the scaffold protein IKKγ/NEMO partly facilitate such functions. As proof-of-principle, we used site-specific mutagenesis to examine the role of NEMO in mediating RANKL-induced signaling in mouse bone marrow macrophages, known as osteoclast precursors. We identified lysine (K)270 as a target regulating RANKL signaling as K270A substitution results in exuberant osteoclastogenesis in vitro and murine inflammatory osteolysis in vivo. Mechanistically, we discovered that K270A mutation disrupts autophagy, stabilizes NEMO, and elevates inflammatory burden. Specifically, K270A directly or indirectly hinders binding of NEMO to ISG15, a ubiquitin-like protein, which we show targets the modified proteins to autophagy-mediated lysosomal degradation. Taken together, our findings suggest that NEMO serves as a toolkit to fine-tune specific signals in physiologic and pathologic conditions.


The human skeleton contains over 200 bones that together act as an internal framework for the body. Over our lifetime, the body constantly removes older bone tissue from the skeleton and replaces it with new bone tissue. This "bone remodeling" also controls how bones are repaired after being damaged by injuries, disease or normal wear and tear. Cells known as osteoclasts are responsible for breaking down old bone tissue and participate in repairing damaged bone. A cellular pathway known as NF-kB signaling stimulates other cells called "bone marrow macrophages" to become osteoclasts. A certain level of NF-kB signaling is required to maintain a healthy skeleton. However, under certain inflammatory conditions, the level of NF-kB signaling becomes too high causing hyperactive osteoclasts to accumulate and inflict severe bone breakdown. This abnormal osteoclast activity leads to eroded and fragile bones and joints, as is the case in diseases such as rheumatoid arthritis and osteoporosis. Previous studies have shown that a protein called NEMO is a core component of the NF-kB signal pathway, but the precise role of NEMO in the diseased response remained unclear. Adapala, Swarnkar, Arra et al. have now used site-directed mutagenesis approach to study the role of NEMO in bone marrow macrophages in mice. The experiments showed that one specific site within the NEMO protein, referred to as lysine 270, is crucial for its role in controlling osteoclasts and the breakdown of bone tissue. Mutating NEMO at lysine 270 led to uncontrolled NF-kB signaling in the bone marrow macrophages. Further experiments showed that lysine 270 served as a sensor to allow NEMO to bind another protein called ISG15, which in turn helped to decrease NF-kB signaling and slow down the erosion of the bone. These findings suggest that site-specific targeting of NEMO, rather than inhibiting the whole NF-kB pathway, may help to reduce the symptoms of bone disease while maintaining the beneficial roles of this essential pathway. However, additional research is required to identify NEMO sites responsible for controlling the inflammatory component.


Subject(s)
Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Osteolysis/metabolism , Animals , Bone Marrow Cells , Gene Expression Regulation , Inflammation/genetics , Intracellular Signaling Peptides and Proteins/genetics , Joint Diseases/metabolism , Joint Diseases/pathology , Mice , Mice, Transgenic , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Osteoclasts/physiology , Osteolysis/genetics , RANK Ligand/genetics , RANK Ligand/metabolism
7.
Int J Mol Sci ; 20(24)2019 Dec 15.
Article in English | MEDLINE | ID: mdl-31847438

ABSTRACT

Patients with gastrointestinal diseases frequently suffer from skeletal abnormality, characterized by reduced bone mineral density, increased fracture risk, and/or joint inflammation. This pathological process is characterized by altered immune cell activity and elevated inflammatory cytokines in the bone marrow microenvironment due to disrupted gut immune response. Gastrointestinal disease is recognized as an immune malfunction driven by multiple factors, including cytokines and signaling molecules. However, the mechanism by which intestinal inflammation magnified by gut-residing actors stimulates bone loss remains to be elucidated. In this article, we discuss the main risk factors potentially contributing to intestinal disease-associated bone loss, and summarize current animal models, illustrating gut-bone axis to bridge the gap between intestinal inflammation and skeletal disease.


Subject(s)
Bone Diseases, Metabolic/pathology , Bone and Bones/pathology , Gastrointestinal Diseases/pathology , Inflammation/pathology , Intestines/pathology , Animals , Humans
8.
Sci Rep ; 9(1): 10429, 2019 07 18.
Article in English | MEDLINE | ID: mdl-31320680

ABSTRACT

Chronic inflammatory insults compromise immune cell responses and ultimately contribute to pathologic outcomes. Clinically, it has been suggested that bone debris and implant particles, such as polymethylmethacrylate (PMMA), which are persistently released following implant surgery evoke heightened immune, inflammatory, and osteolytic responses that contribute to implant failure. However, the precise mechanism underlying this pathologic response remains vague. TREGS, the chief immune-suppressive cells, express the transcription factor Foxp3 and are potent inhibitors of osteoclasts. Using an intra-tibial injection model, we show that PMMA particles abrogate the osteoclast suppressive function of TREGS. Mechanistically, PMMA particles induce TREG instability evident by reduced expression of Foxp3. Importantly, intra-tibial injection of PMMA initiates an acute innate immune and inflammatory response, yet the negative impact on TREGS by PMMA remains persistent. We further show that PMMA enhance TH17 response at the expense of other T effector cells (TEFF), particularly TH1. At the molecular level, gene expression analysis showed that PMMA particles negatively regulate Nrp-1/Foxo3a axis to induce TREG instability, to dampen TREG activity and to promote phenotypic switch of TREGS to TH17 cells. Taken together, inflammatory cues and danger signals, such as bone and implant particles exacerbate inflammatory osteolysis in part through reprogramming TREGS.


Subject(s)
Inflammation/immunology , Neuropilin-1/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Female , Forkhead Transcription Factors/immunology , Immunity, Innate/drug effects , Immunity, Innate/immunology , Male , Mice , Osteoclasts/drug effects , Osteoclasts/immunology , Osteolysis/immunology , Polymethyl Methacrylate , T-Lymphocytes, Regulatory/drug effects , Th17 Cells/drug effects , Th17 Cells/immunology
9.
J Bone Miner Res ; 34(10): 1880-1893, 2019 10.
Article in English | MEDLINE | ID: mdl-31107556

ABSTRACT

Skeletal abnormalities are common comorbidities of inflammatory bowel disease (IBD). Patients suffering from IBD, including ulcerative colitis and Crohn's disease, present with skeletal complications. However, the mechanism underpinning IBD-associated bone loss remains vague. Intestinal inflammation generates an inflammatory milieu at the intestinal epithelium that leads to dysregulation of mucosal immunity through gut-residing innate lymphoid cells (ILCs) and other cell types. ILCs are recently identified mucosal cells considered as the gatekeeper of gut immunity and their function is regulated by intestinal epithelial cell (IEC)-secreted cytokines in response to the inflammatory microenvironment. We first demonstrate that serum as well as IECs collected from the intestine of dextran sulfate sodium (DSS)-induced colitis mice contain high levels of inflammatory and osteoclastogenic cytokines. Mechanistically, heightened inflammatory response of IECs was associated with significant intrinsic activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in IECs and increased frequency of ILC1, ILC3, and myeloid osteoclast progenitors. Validating the central role of IEC-specific NF-κB activation in this phenomenon, conditional expression of constitutively active inhibitor kappa B kinase 2 (IKK2) in IECs in mice recapitulates the majority of the cellular, inflammatory, and osteolytic phenotypes observed in the chemically induced colitis. Furthermore, conditional deletion of IKK2 from IECs significantly attenuated inflammation and bone loss in DSS-induced colitis. Finally, using the DSS-induced colitis model, pharmacologic inhibition of IKK2 was effective in reducing frequency of ILC1 and ILC3 cells, attenuated circulating levels of inflammatory cytokines, and halted colitis-associated bone loss. Our findings identify IKK2 in IECs as viable therapeutic target for colitis-associated osteopenia.


Subject(s)
Bone Resorption/metabolism , Colitis/metabolism , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , NF-kappa B/metabolism , Animals , Bone Resorption/etiology , Bone Resorption/genetics , Bone Resorption/pathology , Colitis/chemically induced , Colitis/complications , Colitis/genetics , Dextran Sulfate/toxicity , Disease Models, Animal , Epithelial Cells/pathology , Female , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Intestinal Mucosa/pathology , Male , Mice , Mice, Knockout , NF-kappa B/genetics
10.
Bone ; 123: 86-91, 2019 06.
Article in English | MEDLINE | ID: mdl-30904629

ABSTRACT

Cytokines and growth factors mediate inflammatory osteolysis in response to particles released from bone implants. However, the mechanism by which this process develops is not entirely clear. Blood vessels and related factors may be required to deliver immune cells and soluble factors to the injury site. Therefore, in the current study we investigated if, vascular endothelial growth factor (VEGF), which is required for angiogenesis, mediates polymethylmethacrylate (PMMA) particles-induced osteolysis. Using bone marrow derived macrophages (BMMs) and ST2 stromal cell line, we show that PMMA particles increase VEGF expression. Further, using a murine calvarial osteolysis model, we found that PMMA injection over calvaria induce significant increase in VEGF expression as well as new vessel formation, represented by von Willebrand factor (vWF) staining. Co-treatment using a VEGF-neutralizing antibody abrogated expression of vWF, indicating decreased angiogenesis. Finally, VEGF neutralizing antibody reduced expression of Tumor necrosis factor (TNF) and decreased osteoclastogenesis induced by PMMA particles in calvariae. This work highlights the significance of angiogenesis, specifically VEGF, as key driver of PMMA particle-induced inflammatory osteolysis, inhibition of which attenuates this response.


Subject(s)
Drug Delivery Systems/methods , Osteolysis/chemically induced , Osteolysis/prevention & control , Polymethyl Methacrylate/toxicity , Skull/drug effects , Vascular Endothelial Growth Factor A/biosynthesis , Animals , Bone Cements/toxicity , Cells, Cultured , Female , Male , Mice , Mice, Inbred C57BL , Microspheres , Osteolysis/metabolism , Random Allocation , Skull/metabolism , Vascular Endothelial Growth Factor A/agonists , Vascular Endothelial Growth Factor A/antagonists & inhibitors
11.
Nucleic Acids Res ; 46(11): 5776-5791, 2018 06 20.
Article in English | MEDLINE | ID: mdl-29373715

ABSTRACT

More than 140 post-transcriptional modifications (PTMs) are known to decorate cellular RNAs, but their incidence, identity and significance in viral RNA are still largely unknown. We have developed an agnostic analytical approach to comprehensively survey PTMs on viral and cellular RNAs. Specifically, we used mass spectrometry to analyze PTMs on total RNA isolated from cells infected with Zika virus, Dengue virus, hepatitis C virus (HCV), poliovirus and human immunodeficiency virus type 1. All five RNA viruses significantly altered global PTM landscapes. Examination of PTM profiles of individual viral genomes isolated by affinity capture revealed a plethora of PTMs on viral RNAs, which far exceeds the handful of well-characterized modifications. Direct comparison of viral epitranscriptomes identified common and virus-specific PTMs. In particular, specific dimethylcytosine modifications were only present in total RNA from virus-infected cells, and in intracellular HCV RNA, and viral RNA from Zika and HCV virions. Moreover, dimethylcytosine abundance during viral infection was modulated by the cellular DEAD-box RNA helicase DDX6. By opening the Pandora's box on viral PTMs, this report presents numerous questions and hypotheses on PTM function and strongly supports PTMs as a new tier of regulation by which RNA viruses subvert the host and evade cellular surveillance systems.


Subject(s)
RNA Processing, Post-Transcriptional , RNA Viruses/genetics , RNA, Viral/metabolism , Cell Line, Tumor , Cytosine/metabolism , DEAD-box RNA Helicases/physiology , Humans , Proto-Oncogene Proteins/physiology , RNA Viruses/metabolism , RNA, Viral/chemistry , Stress, Physiological/genetics
12.
Sci Rep ; 7(1): 12600, 2017 10 03.
Article in English | MEDLINE | ID: mdl-28974699

ABSTRACT

NF-κB signaling is essential for osteoclast differentiation and skeletal homeostasis. We have reported recently that NUMB-like (NUMBL) protein modulates osteoclastogenesis by down regulating NF-κB activation. Herein, we decipher the mechanism underlying this phenomenon. We found that whereas NUMBL mRNA expression decreases upon stimulation of wild type (WT) bone marrow macrophages (BMMs) with RANKL, TAK1 deficiency in these cells leads to increased NUMBL and decreased TRAF6 and NEMO expression. These changes were restored upon WT-TAK1 expression, but not with catalytically inactive TAK1-K63W, suggesting that TAK1 enzymatic activity is required for these events. Forced expression of NUMBL inhibits osteoclast differentiation and function as evident by reduction in all hallmarks of osteoclastogenesis. Conversely, NUMBL-null BMMs, show increased osteoclast differentiation and mRNA expression of osteoclast marker genes. Post-translationally, K48-linked poly-ubiquitination of NUMBL is diminished in TAK1-null BMMs compared to elevated K48-poly-ubiquitination in WT cells, indicating increased stability of NUMBL in TAK1-null conditions. Further, our studies show that NUMBL directly interacts with TRAF6 and NEMO, and induces their K48-poly-ubiquitination mediated proteasomal degradation. Collectively, our data suggest that NUMBL and TAK1 are reciprocally regulated and that NUMBL acts as an endogenous regulator of NF-κB signaling and osteoclastogenesis by targeting the TAK1-TRAF6-NEMO axis.


Subject(s)
Intracellular Signaling Peptides and Proteins/genetics , Nerve Tissue Proteins/genetics , Nuclear Receptor Subfamily 2, Group C, Member 2/genetics , Osteogenesis/genetics , TNF Receptor-Associated Factor 6/genetics , Animals , Cell Differentiation/genetics , Gene Expression Regulation, Developmental/genetics , Mice , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , NF-kappa B/genetics , Osteoclasts/metabolism , Proteasome Endopeptidase Complex/genetics , RNA, Messenger/genetics , Signal Transduction
13.
PLoS One ; 9(3): e93108, 2014.
Article in English | MEDLINE | ID: mdl-24667334

ABSTRACT

AIDS is a global pandemic that has seen the development of novel and effective treatments to improve the quality of life of those infected and reduction of spread of the disease. Palmitic Acid (PA), which we identified and isolated from Sargassum fusiforme, is a naturally occurring fatty acid that specifically inhibits HIV entry by binding to a novel pocket on the CD4 receptor. We also identified a structural analogue, 2-bromopalmitate (2-BP), as a more effective HIV entry inhibitor with a 20-fold increase in efficacy. We have used the structure-activity relationship (SAR) of 2-BP as a platform to identify new small chemical molecules that fit into the various identified active sites in an effort to identify more potent CD4 entry inhibitors. To validate further drug development, we tested the PA and 2-BP scaffold molecules for genotoxic potential. The FDA and International Conference on Harmonisation (ICH) recommends using a standardized 3-test battery for testing compound genotoxicity consisting of the bacterial reverse mutation assay, mouse lymphoma assay, and rat micronucleus assay. PA and 2-BP and their metabolites tested negative in all three genotoxicty tests. 2-BP is the first derivative of PA to undergo pre-clinical screening, which will enable us to now test multiple simultaneous small chemical structures based on activity in scaffold modeling across the dimension of pre-clinical testing to enable transition to human testing.


Subject(s)
Biological Products/chemistry , Biological Products/toxicity , HIV Fusion Inhibitors/chemistry , HIV Fusion Inhibitors/toxicity , HIV/drug effects , HIV/physiology , Virus Internalization/drug effects , Animals , Biological Products/pharmacology , Drug Discovery , Female , HIV Fusion Inhibitors/pharmacology , Lymphoma/pathology , Male , Mice , Micronucleus Tests , Palmitates/chemistry , Palmitates/pharmacology , Palmitates/toxicity , Palmitic Acid/chemistry , Palmitic Acid/pharmacology , Palmitic Acid/toxicity , Rats , Salmonella typhimurium/drug effects , Salmonella typhimurium/genetics , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...