Systemic inflammatory cytokine profiles in patients with gout during flare, intercritical and treat-to-target phases: TNFSF14 as new biomarker.

INTRODUCTION: Untreated gout is characterised by monosodium urate (MSU) crystal accumulation responsible for recurrent flares that are commonly separated by asymptomatic phases. Both phases are inflammatory conditions of variable intensity. Gout flares are self-limited inflammatory reactions involving multiple mediators. This study aimed to characterise the inflammatory profiles of gout at different phases. METHODS: Using the Olink targeted proteomics, levels of 92 inflammation-related proteins were measured in plasma samples of a prospective gout population (GOUTROS), collected at gout flare (T1), the intercritical phase (T2) and after reaching the target serum urate level under urate-lowering therapy (T3). Results were validated in an independent cohort (OLT1177-05) with plasmas collected at T1 and T2. Ex vivo and in vitro experiments were performed to assess the inflammatory properties of new biomarkers. RESULTS: In total, 21 inflammatory new biomarkers were differentially expressed during the three time-points of gout disease. The levels of four of these proteins (interleukin 6 (IL-6), colony-stimulating factor 1, vascular endothelial growth factor A and tumour necrosis factor superfamily 14 (TNFSF14)) were increased during gout flare in an independent cohort. IL-6 and TNFSF14 had the highest fold change in expression during T1 versus T2 or T3. TNFSF14 was produced at the inflamed joint and enhanced the inflammatory response induced by lipopolysaccharide and MSU crystal stimulation. Conversely, TNFSF14 blockade reduced the inflammatory response. Additionally, single nucleotide polymorphisms of TNFSF14 affected the ability of myeloid cells to produce inflammatory cytokines. CONCLUSION: Gout flare involves multiple inflammatory mediators that may be used as potential therapeutic targets.

Interactions between cadmium and zinc on gene expression pattern of differentiation markers in MC3T3-E1 cell line.

We evaluated, in vitro, the interactions between cadmium (Cd) and zinc (Zn) during the proliferation and differentiation process using bone MC3T3-E1 cell line.Cells were treated with CdCl2 and/or ZnCl2 for 24 and 48 h and 5 µM CdCl2 was found as low cytotoxic dose and 25 µM ZnCl2 as the best Zn treatment for cell proliferation. Gene expression of some bone markers (Runx2, collagen α1 (Colα1), osteocalcin (Oc), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) was studied at 24, 48 and 72 h.Treatment by CdCl2 depressed Runx2, Colα1, and BSP mRNA levels after 24 h. Oc and ALP gene expression was found to be decreased after 72 h.CdCl2 -exposure decreased ALP activity and Ca deposit in matrix. In concomitant treatment by CdCl2 and ZnCl2, gene expression of osteoblastic markers was found to be up-regulated (p < 0, 05) compared to CdCl2 treated cells, ALP staining and mineralization were increased.Our results show that Zn could prevent Cd-induced toxicity on MC3T3-E1 cells, probably through the restoration of Runx2, col α1, BSP, ALP and Oc and gene expression inhibited by Cd.

Effect of Rubus idaeus Extracts in Murine Chondrocytes and Explants.

Osteoarthritis is characterized by cartilage loss resulting from the activation of chondrocytes associated with a synovial inflammation. Activated chondrocytes promote an increased secretion of matrix proteases and proinflammatory cytokines leading to cartilage breakdown. Since natural products possess anti-inflammatory properties, we investigated the direct effect of Rubus idaeus extracts (RIE) in chondrocyte metabolism and cartilage loss. The effect of RIE in chondrocyte metabolism was analyzed in murine primary chondrocytes and cartilage explants. We also assessed the contribution of RIE in an inflammation environment by culturing mice primary chondrocytes with the supernatant of Raw 264.7 macrophage-like cells primed with RIE. In primary chondrocytes, RIE diminished chondrocyte hypertrophy (Col10), while increasing the expression of catabolic genes (Mmp-3, Mmp-13) and reducing anabolic genes (Col2a1, Acan). In cartilage explants, Rubus idaeus prevented the loss of proteoglycan (14.84 ± 3.07% loss of proteoglycans with IL1 alone vs. 3.03 ± 1.86% with IL1 and 100 µg/mL of RIE), as well as the NITEGE neoepitope expression. RIE alone reduced the expression of Il1 and Il6 in macrophages, without changes in Tnf and Cox2 expression. The secretome of macrophages pre-treated with RIE and transferred to chondrocytes decreases the gene and protein expression of Mmp-3 and Cox2. In conclusion, these data suggest that RIE may protect from chondrocyte catabolism and cartilage loss in inflammatory conditions. Further evaluations are need before considering RIE as a candidate for the treatment for osteoarthritis.

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-ß-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-ß-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-ß-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis.

Increased NF-κB Activity and Decreased Wnt/ß-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice.

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased ß-catenin phosphorylation, reduced osteoblast ß-catenin expression, and altered expression of Wnt/ß-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/ß-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/ß-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.