NAC is a thiolic antioxidant produced by the body and serves as a precursor of glutathione synthesis. In rheumatoid arthritis (RA), oxidative stress is an important mechanism causing destructive proliferative synovitis.
Objectives
This study aimed to determine the regulatory role of N-Acetyl-L-cysteine (NAC), an antioxidant, in IL-17-induced osteoclast differentiation in RA.
Methods
After RA synovial fibroblasts were stimulated by IL-17, the expression and production of RANKL was determined by real-time PCR and ELISA. Human peripheral blood monocytes were cultured with M-CSF, IL-17, RANKL, and/or various concentrations of NAC, followed by counting of the cells for tartrate-resistant acid phosphatase activity to determine osteoclast formation. Osteoclastogenesis was also determined after cocultures of IL-17-stimulated RA synovial fibroblasts, Th17 cells and various concentrations of NAC with monocytes. After human peripheral CD4+ T cells were cultured with NAC under Th17 condition, IL-17, IFN-g, IL-4, Foxp3, RANKL and IL-2 expression and production was determined by flow cytometry or ELISA.
Results
When RA synovial fibroblasts were stimulated by IL-17, IL-17 stimulated the production of RANKL, and NAC reduced the IL-17-induced RANKL production in a dose-dependent manner. NAC decreased IL-17-activated phosphorylation of mTOR, JNK and IkB. When human peripheral blood CD14+ monocytes were cultured with M-CSF and IL-17 or RANKL, osteoclasts were differentiated, and NAC reduced the osteoclastogenesis. After human peripheral CD4+ T cells were co-cultured with IL-17-pretreated RA synovial fibroblasts or Th17 cells, NAC reduced their osteoclastogenesis. Under Th17 polarizing condition, NAC decreased Th17 cell differentiation and IL-17 and RANKL production.
Conclusions
NAC inhibits the IL-17-induced RANKL production in RA synovial fibroblasts and IL-17-induced osteoclast differentiation. NAC also reduced Th17 polarization. NAC could be a supplementary therapeutic option for inflammatory and bony destructive processes in RA.
Acknowledgements
This research was supported by a grant of the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea (NRF-2014R1A2A2A01007223) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI13C1704).
1131-Plat Single Molecule Diagnostic Method to Reveal Cancer-Related EGFR Signaling Hong-Won Lee, Min Kwon Cha, Kihyuk Shin, Seung-Hyo Lee, Tae-Young Yoon. KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon, Korea, Republic of. Receptor tyrosine kinases (RTK) regulate cell fate such as differentiation, proliferation, survival and migration via recruiting SH2 containing downstream proteins. Dysregulated interaction between RTK and SH2 containing downstream protein incessantly activates MAPK or PI3K-Akt pathway which will cause a cancer in various organs. Real-time single-molecule coimmunoprecipitation (co-IP) is able to reveal quantitatively these proteinprotein interactions using endogenous proteins at single molecule resolution. Here we develop a single molecule diagnostic method measuring endogenous epidermal growth factor receptor (EGFR) signaling extracted from cancer cell lines or lung tissues of cancer patients with eGFP labeled Grb2 as a probe, an adaptor protein containing SH2 involved in various EGFR signaling pathways using real-time single-molecule co-IP. This method distinguishes cell lines or tissues expressing highly activated EGFR. We also demonstrate that even cell lines have the same EGFR mutation, aA746-E750, protein interaction network can be altered, which is not revealed by genome sequencing based diagnosis. Our approach allows us to investigate signaling network of proteins at single molecule resolution. It also suggests a concept of a molecular diagnostic method at protein-protein interaction level.
Protein-cage nanoparticles are promising multifunctional platforms for targeted delivery of imaging and therapeutic agents owing to their biocompatibility, biodegradability, and low toxicity. The major advantage of protein-cage nanoparticles is the ability to decorate their surfaces with multiple functionalities through genetic and chemical modification to achieve desired properties for therapeutic and/or diagnostic purposes. Specific peptides identified by phage display can be genetically fused onto the surface of cage proteins to promote the association of nanoparticles with a particular cell type or tissue. Upon symmetrical assembly of the cage, peptides are clustered on the surface of the cage protein in bunches. The resulting PBNC (peptide bunches on nanocage) offers the potential of synergistically increasing the avidity of the peptide ligands, thereby enhancing their blocking ability for therapeutic purposes. Here, we demonstrated a proof-of-principle of PBNCs, fusing the interleukin-4 receptor (IL-4R)-targeting peptide, AP-1, identified previously by phage display, with ferritin-L-chain (FTL), which undergoes 24-subunit assembly to form highly stable AP-1-containing nanocage proteins (AP1-PBNCs). AP1-PBNCs bound specifically to the IL-4R-expressing cell line, A549, and their binding and internalization were specifically blocked by anti-IL-4R antibody. AP1-PBNCs exhibited dramatically enhanced binding avidity to IL-4R compared with AP-1 peptide, measured by surface plasmon resonance spectroscopy. Furthermore, treatment with AP1-PBNCs in a murine model of experimental asthma diminished airway hyper-responsiveness and eosinophilic airway inflammation along with decreased mucus hyperproduction. These findings hold great promise for the application of various PBNCs with ligand-specific peptides in therapeutics for different diseases, such as cancer.
In this review, we will explore the intricate roles of cytokines and vascular endothelial growth factors in autoimmune diseases (ADs), with a particular focus on rheumatoid arthritis (RA) and multiple sclerosis (MS). AD is characterized by self-destructive immune responses due to auto-reactive T lymphocytes and Abs. Among various types of ADs, RA and MS possess inflammation as a central role but in different sites of the patients. Other common aspects among these two ADs are their chronicity and relapsing-remitting symptoms requiring continuous management. First factor inducing these ADs are cytokines, such as IL-6, TNF-α, and IL-17, which play significant roles in the pathogenesis by contributing to inflammation, immune cell activation, and tissue damage. Secondly, vascular endothelial growth factors, including VEGF and angiopoietins, are crucial in promoting angiogenesis and inflammation in these two ADs. Finally, placental growth factor (PlGF), an emerging factor with bi-directional roles in angiogenesis and T cell differentiation, as we introduce as an "angio-lymphokine" is another key factor in ADs. Thus, while angiogenesis recruits more inflammatory cells into the peripheral sites, cytokines secreted by effector cells play critical roles in the pathogenesis of ADs. Various therapeutic interventions targeting these soluble molecules have shown promise in managing autoimmune pathogenic conditions. However, delicate interplay between cytokines, angiogenic factors, and PlGF has more to be studied when considering their complementary role in actual pathogenic conditions. Understanding the complex interactions among these factors provides valuable insights for the development of innovative therapies for RA and MS, offering hope for improved patient outcomes.
BackgroundTobacco-related lung diseases, including chronic obstructive pulmonary disease (COPD), are major causes of lung-related disability and death worldwide. Acute exacerbation of COPD (AE-COPD) is commonly associated with upper and lower respiratory tract viral infections and can result in respiratory failure in those with advanced lung disease.ObjectiveWe sought to determine the mechanism underlying COPD exacerbation and host response to pathogen-derived factors.MethodsOver a 24-month period, we assessed the viral causes for upper and lower respiratory tract infections in patients with COPD (n = 155) and control subjects (n = 103). We collected nasal and bronchoalveolar lavage fluid and peripheral blood under baseline and exacerbated conditions. We determined the effect of human rhinovirus (HRV) proteinases on T-cell activation in human subjects and mice.ResultsHRVs are isolated from nasal and lung fluid from subjects with AE-COPD. Bronchoalveolar lavage fluid and CD4 T cells from patients with COPD exhibited a TH1 and TH2 cell cytokine phenotype during acute infection. HRV-encoded proteinase 2A activated monocyte-derived dendritic cells in vitro and induced strong TH1 and TH2 immune responses from CD4 T cells. Intranasal administration of recombinant rhinovirus proteinase 2A in mice resulted in an increase in airway hyperreactivity, lung inflammation, and IL-4 and IFN-γ production from CD4 T cells.ConclusionOur findings suggest that patients with severe COPD show TH1- and TH2-biased responses during AE-COPD. HRV-encoded proteinase 2A, like other microbial proteinases, could provide a TH1- and TH2-biasing adjuvant factor during upper and lower respiratory tract infection in patients with severe COPD. Alteration of the immune response to secreted viral proteinases might contribute to worsening of dyspnea and respiratory failure in patients with COPD. Tobacco-related lung diseases, including chronic obstructive pulmonary disease (COPD), are major causes of lung-related disability and death worldwide. Acute exacerbation of COPD (AE-COPD) is commonly associated with upper and lower respiratory tract viral infections and can result in respiratory failure in those with advanced lung disease. We sought to determine the mechanism underlying COPD exacerbation and host response to pathogen-derived factors. Over a 24-month period, we assessed the viral causes for upper and lower respiratory tract infections in patients with COPD (n = 155) and control subjects (n = 103). We collected nasal and bronchoalveolar lavage fluid and peripheral blood under baseline and exacerbated conditions. We determined the effect of human rhinovirus (HRV) proteinases on T-cell activation in human subjects and mice. HRVs are isolated from nasal and lung fluid from subjects with AE-COPD. Bronchoalveolar lavage fluid and CD4 T cells from patients with COPD exhibited a TH1 and TH2 cell cytokine phenotype during acute infection. HRV-encoded proteinase 2A activated monocyte-derived dendritic cells in vitro and induced strong TH1 and TH2 immune responses from CD4 T cells. Intranasal administration of recombinant rhinovirus proteinase 2A in mice resulted in an increase in airway hyperreactivity, lung inflammation, and IL-4 and IFN-γ production from CD4 T cells. Our findings suggest that patients with severe COPD show TH1- and TH2-biased responses during AE-COPD. HRV-encoded proteinase 2A, like other microbial proteinases, could provide a TH1- and TH2-biasing adjuvant factor during upper and lower respiratory tract infection in patients with severe COPD. Alteration of the immune response to secreted viral proteinases might contribute to worsening of dyspnea and respiratory failure in patients with COPD.
To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL ( Fasl gld/gld )- and soluble FasL ( Fasl Δs/Δs )-deficient mice, but not in Fas ( Fas lpr/lpr and Fas –/– )- or membrane FasL ( Fasl Δm/Δm )-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL–Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL–DR5 interaction-mediated CX3CL1–CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL–DR5 interaction promotes inflammation and is a potential therapeutic target.
Abstract Background Many studies have found that smoking reduces lung function, but the relationship between cigarette smoke and allergic asthma has not been clearly elucidated, particularly the role of mast cells. This study aimed to investigate the effects of smoke exposure on allergic asthma and its association with mast cells. Methods BALB/c mice were sensitized and challenged by OVA to induce asthma, and bone marrow-derived mast cells (BMMCs) were stimulated with antigen/antibody reaction. Mice or BMMCs were exposed to cigarette smoke or CSE solution for 1 mo or 6 h, respectively. The recruitment of inflammatory cells into BAL fluid or lung tissues was determined by Diff-Quik or H&E staining, collagen deposition by Sircol assay, penh values by a whole-body plethysmography, co-localization of tryptase and Smad3 by immunohistochemistry, IgE and TGF-β level by ELISA, expressions of Smads proteins, activities of signaling molecules, or TGF-β mRNA by immunoblotting and RT-PCR. Results Cigarette smoke enhanced OVA-specific IgE levels, penh values, recruitment of inflammatory cells including mast cells, expressions of smad family, TGF-β mRNA and proteins, and cytokines, phosphorylations of Smad2 and 3, and MAP kinases, co-localization of tryptase and Smad3, and collagen deposition more than those of BAL cells and lung tissues of OVA-induced allergic mice. CSE solution pretreatment enhanced expressions of TGF-β, Smad3, activities of MAP kinases, NF-κB/AP-1 or PAI-1 more than those of activated-BMMCs. Conclusions The data suggest that smoke exposure enhances antigen-induced mast cell activation via TGF-β/Smad signaling pathways in mouse allergic asthma, and that it exacerbates airway inflammation and remodeling.
Objectives: Elastin is a major structural protein of arteries and elastin derived peptide is known to be related to arterial change. We previously reported a novel assay for anti-aortic elastin antibody, but its clinical implication has not been clearly shown. The aim of this study was to check if anti-aortic elastin antibody titers may reflect the risk of coronary artery disease (CAD) or its detail characteristics. Methods: This study included 174 CAD patients and 171 age-, sex-matched control subjects. In all subjects, anti-aortic elastin antibody titer was quantified by ELISA. Parameters of arterial stiffness including augmentation index (AI) and heart to femoral pulse wave velocity (hfPWV) were measured non-invasively. In patients with CAD, clinical and angiographic characteristics were evaluated. Associations between anti-aortic elastin and vascular characteristics were identified by linear regression analysis. Results: Median blood level of anti-aortic elastin was significantly lower in the CAD group than that of the control group (197 a.u. vs. 63 a.u., p<0.001). Levels of anti-aortic elastin were significantly lower in males, subjects with hypertension, diabetes mellitus, hyperlipidemia, or hfPWV (Figure). However, the levels were not dependent of atherothrombotic events or angiographic severity of CAD (Figure). In multivariate analysis, male (β=-0.38, p<0.001), diabetes mellitus (β=-0.62, p<0.001), hyperlipidemia (β=-0.29, p<0.001), and AI (β=-0.006, p=0.02) were finally identified as determinants for anti-aortic elastin levels (Table). Conclusions: Taken together, lower levels of anti-aortic elastin are related to CAD. The association between antibody titer and CAD is linked to arterial stiffness rather than advancement of atherosclerosis.
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