Immunoprofiling of active and inactive systemic juvenile idiopathic arthritis reveals distinct biomarkers: a single-center study.

BACKGROUND: This study aimed to perform an immunoprofiling of systemic juvenile idiopathic arthritis (sJIA) in order to define biomarkers of clinical use as well as reveal new immune mechanisms. METHODS: Immunoprofiling of plasma samples from a clinically well-described cohort consisting of 21 sJIA patients as well as 60 age and sex matched healthy controls, was performed by a highly sensitive proteomic immunoassay. Based on the biomarkers being significantly up- or down-regulated in cross-sectional and paired analysis, related canonical pathways and cellular functions were explored by Ingenuity Pathway Analysis (IPA). RESULTS: The well-studied sJIA biomarkers, IL6, IL18 and S100A12, were confirmed to be increased during active sJIA as compared to healthy controls. IL18 was the only factor found to be increased during inactive sJIA as compared to healthy controls. Novel factors, including CASP8, CCL23, CD6, CXCL1, CXCL11, CXCL5, EIF4EBP1, KITLG, MMP1, OSM, SIRT2, SULT1A1 and TNFSF11, were found to be differentially expressed in active and/or inactive sJIA and healthy controls. No significant pathway activation could be predicted based on the limited factor input to the IPA. High Mobility Group Box 1 (HMGB1), a damage associated molecular pattern being involved in a series of inflammatory diseases, was determined to be higher in active sJIA than inactive sJIA. CONCLUSIONS: We could identify a novel set of biomarkers distinguishing active sJIA from inactive sJIA or healthy controls. Our findings enable a better understanding of the immune mechanisms active in sJIA and aid the development of future diagnostic and therapeutic strategies.

Cleavage of HMGB1 by Proteolytic Enzymes Associated with Inflammatory Conditions.

Extracellular HMGB1 acts as an alarmin in multiple autoimmune diseases. While its release and functions have been extensively studied, there is a substantial lack of knowledge regarding HMGB1 regulation at the site of inflammation. Herein we show that enzymes present in arthritis-affected joints process HMGB1 into smaller peptides in vitro. Gel electrophoresis, Western blotting and mass spectrometry analyses indicate cleavage sites for human neutrophil elastase, cathepsin G, and matrix metalloproteinase 3 within the HMGB1 structure. While human neutrophil elastase and matrix metalloproteinase 3 might alter the affinity of HMGB1 to its receptors by cleaving the acidic C-terminal tail, cathepsin G rapidly and completely degraded the alarmin. Contrary to a previous report we demonstrate that HMGB1 is not a substrate for dipeptidyl peptidase IV. We also provide novel information regarding the presence of these proteases in synovial fluid of juvenile idiopathic arthritis patients. Correlation analysis of protease levels and HMGB1 levels in synovial fluid samples did not, however, reveal any direct relationship between the recorded levels. This study provides knowledge of proteolytic processing of HMGB1 relevant for the regulation of HMGB1 during inflammatory disease.

Effect of Aging on Taurine Transporter (TauT) Expression in the Mouse Brain Cortex.

Taurine content in an older brain is decreased compared to a younger brain and is associated with cognitive deficits. It is not yet known whether the decrease in taurine content is associated with decreased expression of taurine inflow mediating transporters during the aging process. In this study, we investigated whether aging affects taurine transporter and glycine transporter 1 expression in the brain cortex of the mouse. Taurine and glycine transporter expression was compared in the brain cortex of C57BL/6 mice at different ages (2, 12, and 24 months) and to age-matched NLRP3 inflammasome knockout mice. In wild type mice, taurine transporter (TauT) expression in the brain cortex of 12- or 24-month-old mice did not significantly differ from TauT expression in 2-month-old mice. Moreover, TauT expression in the brain cortex of 12- or 24-month-old mice did not significantly differ from age-matched NLRP3 KO mice. This result indirectly suggests that TauT expression may be not affected by aging or age-induced inflammation. In addition, glycine transporter expression was similar to the TauT expression pattern. In conclusion, aging and age-related inflammation might not significantly affect taurine and glycine transporter expression in aged mice. Thus, the decrease of taurine content in an older brain, which is associated with cognitive deficits, may not be significantly related to altered taurine and glycine transporter expression.

Effects of Chronic Intake of a Low Concentration of Taurine on Physical Strength and Body Composition in Mice.

Most studies of taurine on athletic performance have been conducted at acute and high doses in rodents. These doses and duration of administration are not reasonable for normal human life. Thus, it is not valid to extrapolate these animal results to people. Dose and duration that mimic human use of taurine in normal life can help to clarify the taurine effect in humans. This study investigated whether long-term, low-dose taurine (2% taurine drinking water for 25 weeks), similar to normal taurine intake in humans, can affect endurance exercise and body composition. Twenty ICR mice were divided into two groups. The control group received normal drinking water, and the taurine treated group received 2% taurine drinking water for 25 weeks. The mice were evaluated for body composition by mass and for physical strength by treadmill exhaustion and suspension tests. The supply of chronic 2% taurine drinking water has a slight effect on weight gain. In body composition analysis, a slight increase in body weight was due to an increase in muscle mass, not an increase in body fat. However, taurine ingestion did not increase endurance exercise. In conclusion, these results indirectly suggest that acute, high-dose taurine treatment is better than long-term, low-dose treatment to increase athletic performance.

Guggulipid ameliorates adjuvant-induced arthritis and liver oxidative damage by suppressing inflammatory and oxidative stress mediators.

BACKGROUND: Arthritis is a common degenerative joint disease characterized by deterioration of articular cartilage, subchondral bone, and associated with immobility, pain and inflammation. The incessant action of reactive oxygen species (ROS) during progressive arthritis causes severe oxidative damage to vital organs and circulatory system. PURPOSE: In this study we investigated the ability of guggulipid (GL), a lipid rich extract from the gum resin of the plant Commiphora whighitii to suppress the progressive arthritis and associated liver oxidative stress both in vivo and in vitro. STUDY DESIGN/METHODS: The anti-arthritic ability of GL was demonstrated in vitro using IL-1ß stimulated bovine nasal cartilage model and in vivo Freund's complete adjuvant-induced arthritic rat model. Collagen/proteoglycan degradation and pro-inflammatory mediators were monitored in the harvested culture medium of nasal cartilage by estimating the levels of matrix metalloproteinases (MMPs), hydroxy proline, glycosaminoglycans and inflammatory mediators. Further, anti-arthritic ability of GL was evaluated in vivo by measuring enzymatic and non-enzymatic mediators of cartilage degradation, inflammation and oxidative stress markers. RESULTS: GL significantly inhibited the IL-1ß stimulated cartilage degradation in vitro by mitigating the MMPs activity, collagen degradation and secretion of pro-inflammatory mediators. Further, GL significantly reduced the adjuvant-induced paw swelling and body weight loss in vivo. GL remarkably reduced the MMPs and hyaluronidases activities in serum and bone homogenate along with altered hematological parameters. GL also mitigated the elevated bone resorbing enzymes cathepsins, exoglycosidases and phosphatases. Additionally, GL effectively mitigated ROS and oxidative stress-mediators recuperating the altered serum/liver oxidative stress and liver damage incurred during arthritic progression. CONCLUSION: In summary, the study clearly demonstrates the protective efficacy of GL against arthritis and its associated oxidative stress, particularly, liver oxidative damage. Hence, GL could be a potential alternative and complementary medicine to treat inflammatory joint diseases.

Targeted delivery of p-coumaric acid encapsulated mannosylated liposomes to the synovial macrophages inhibits osteoclast formation and bone resorption in the rheumatoid arthritis animal model.

The current study aimed to target the delivery of p-coumaric acid (CA), a dietary polyphenol to the synovial macrophages of AIA rats via mannose incorporated liposomal delivery system (ML) with reference to osteoclastogenesis and bone resorption. In vivo imaging and in vitro drug release study indicated the efficiency of mannosylated liposomes to localize at the site of inflammation and increased sustain drug release respectively. Morphological assessment of isolated synovial macrophages with respect to CD86 (synovial macrophages) and CD51 (pre-/osteoclast) indicated that p-coumaric acid encapsulated mannosylated liposomes (ML-CA) inhibited the osteoclasts differentiation. ML-CA treatment inhibited the TRAP staining, downregulated the expression of MMP-9 and NFATc1 and inflammatory cytokines. The ex-vivo study specified the ability of CA to induce the OPG production in bone marrow stromal cell triggered macrophage-osteoclasts differentiation and to preserve the calcium content. Taken together, our results demonstrated that ML-CA could intervene in the osteoclast formation.

Targeting RAW 264.7 macrophages (M1 type) with Withaferin-A decorated mannosylated liposomes induces repolarization via downregulation of NF-κB and controlled elevation of STAT-3.

In the present study, we intend to gain an insight into the mechanism of Withaferin-A (WA), a steroidal lactone with reference to repolarization of RAW 264.7 macrophages (M1 to M2 type). We found that successful internalization of WA via mannosylated liposomal delivery system (ML-WA) reduced the RAW 264.7 macrophage (M1) mediated pro-inflammatory cytokines (IL-1ß, IL-6, IL-23, and TNF-α) through the attenuation of transcription factor NF-κB-p65 expression. Whereas, ML-WA treatment induced a controlled upregulation of p-STAT3, and ablated the key oxidative stress markers (NO, iNOS, and ROS) in M1 → M2 RAW 264.7 macrophage repolarization, which suggested the recalibration of M1 macrophage metabolic function. Further, the elevated expression of M2 macrophage associated CD163 over the M1 macrophage related CD86 concluded that ML-WA induces an anti-inflammatory response by repolarizing the M1 → M2 RAW 264.7 macrophage.

p-Coumaric acid, a dietary polyphenol ameliorates inflammation and curtails cartilage and bone erosion in the rheumatoid arthritis rat model.

This study was designed to explore the underlying mechanism of p-coumaric acid (CA), a dietary polyphenol in adjuvant-induced arthritis (AIA) rat model with reference to synovitis and osteoclastogenesis. Celecoxib (COX-2 selective inhibitor) (5 mg/kg b.wt) was used as a reference drug. CA remarkably suppressed the paw edema, body weight loss and inflammatory cytokine and chemokine levels (TNF-α, IL-1ß, IL-6, and MCP-1) in serum and ankle joint of arthritic rats. Consistently, CA reduced the expression of osteoclastogenic factors (RANKL and TRAP), pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-17), and inflammatory enzymes (iNOS and COX-2) in arthritic rats. However, OPG expression was found elevated. Besides, the abundance of transcription factors (NF-κB-p65, and p-NF-κB-p65, NFATc-1, and c-Fos) and MAP kinases (JNK, p-JNK, and ERK1/2) expression was alleviated in CA administered arthritic rats. In addition, CA truncated osteoclastogenesis by regulating the RANKL/OPG imbalance in arthritic rats and suppressing the RANKL-induced NFATc-1 and c-Fos expression in vitro. Radiological (CT and DEXA scan) and histological assessments authenticated that CA inhibited TRAP, bone destruction and cartilage degradation in association with enhanced bone mineral density. Taken together, our findings suggest that CA demonstrated promising anti-arthritic effect and could prove useful as an alternative drug in RA therapeutics. © 2017 BioFactors, 43(5):698-717, 2017.

Withaferin-A, a steroidal lactone encapsulated mannose decorated liposomes ameliorates rheumatoid arthritis by intriguing the macrophage repolarization in adjuvant-induced arthritic rats.

In order to develop a better therapeutic approach for the treatment of rheumatoid arthritis (RA), withaferin-A; a steroidal lactone incorporated with mannosylated liposomes (ML-WA) was administered to adjuvant induced arthritic rats in intent to target the synovial macrophages. The confocal microscopy studies showed a successful internalization of ML-WA in the primarily isolated synovial macrophages. Consequently, targeting synovial macrophages via ML-WA reduced the oxidative stress (ROS and NO), and paw edema, however, a progressive gain in the body weight was observed in AIA rats. ML-WA treatment upregulated the production of osteoprotegerin (OPG) and downregulated the release of receptor activator of nuclear factor-κB ligand (RANKL), favoring osteoclastogenesis negatively. Correspondingly, the ankle joints were found intact with no bone erosion and cartilage degradation in ML-WA treated AIA rats as evidenced by histopathological analysis. Also, synovial macrophage assessment showed that the concentration and the gene amplification of M1 macrophage mediated pro-inflammatory mediators (TNF-α, IL-1ß, IL-6, MCP-1 and VEGF) were curtailed in ML-WA treated AIA rats. In contrast, anti-inflammatory cytokine (IL-10) was found abundantly released. Furthermore, the mRNA expression of the M1 surface marker (CD86) was found down regulated, whereas, M2 marker (CD163) was highly amplified in ML-WA treated synovial macrophages of arthritic rats. Cumulatively, our result signified that targeted delivery of ML-WA ameliorated the severity of inflammation and bone resorption in AIA rats via M1 to M2 macrophage repolarization.

Targeted delivery of morin, a dietary bioflavanol encapsulated mannosylated liposomes to the macrophages of adjuvant-induced arthritis rats inhibits inflammatory immune response and osteoclastogenesis.

The purpose of the study was to develop a liposomal drug delivery system for morin, a dietary polyphenol, in order to target the synovial macrophages and investigate the remission of disease severity in the adjuvant-induced arthritic (AIA) rats. To do so, mannose decorated liposomal morin (ML-Morin) was prepared using the thin film hydration method and the physicochemical properties were characterized. The particle size and zeta potential of liposomal morin (L-Morin) was found to be 127.9nm±2.6 and -24.5mV±0.76, whereas ML-Morin showed an increased value of 132.5nm±5.2 and -54.8mV±0.67 respectively. Further, the drug entrapment efficiency (% EE) of ML-Morin was found 86.7±3.8%. To understand the efficacy of L-Morin, ML-Morin over free-Morin; cellular uptake, production and expression of pro-inflammatory mediators, osteoclastogenic factors, and transcription factors were evaluated in primarily isolated synovial and spleen macrophages. Interestingly, confocal microscopic images showed an increased uptake of ML-Morin in the synovial and spleen macrophages than L-morin. In addition, ML-Morin significantly suppressed the production and mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-17), angiogenic factors (VEGF), an inflammatory enzyme (iNOS), and transcription factor (NF-κB-p65). Furthermore, the protein expression of TNF-α, IL-1ß, IL-6, IL-17, RANKL, STAT-3, and p-STAT-3 was found to decrease with increased osteoprotegerin (OPG) expression in the ML-Morin targeted macrophages. Thus, our findings endorsed that, ML-Morin preferential internalization into the macrophages of arthritic rats effectively inhibited the inflammatory immune response and osteoclastogenesis better than the dexamethasone palmitate encapsulated mannosylated liposomes (ML-DP), a reference drug as evidenced by clinical and histological analysis.