Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis

The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of small nucleolar RNAs (snoRNAs) in joint ageing and OA may provide diagnostic biomarkers and therapeutic targets. This study determined expression patterns of snoRNAs in joint ageing and OA and examined them as potential biomarkers. Using SnoRNASeq and real-time quantitative PCR (qRT-PCR) we demonstrate snoRNA expression levels in murine ageing and OA joints and serum for the first time. SnoRNASeq identified differential expression (DE) of 6 snoRNAs in young versus old joints and 5 snoRNAs in old sham versus old experimental osteoarthritic joints. In serum we found differential presence of 27 snoRNAs in young versus old serum and 18 snoRNAs in old sham versus old experimental osteoarthritic serum. Confirmatory qRT-PCR analysis demonstrated good correlation with SnoRNASeq findings. Profiling the expression patterns of snoRNAs is the initial step in determining their functional significance in ageing and osteoarthritis, and provides potential diagnostic biomarkers and therapeutic targets. Our results establish snoRNAs as novel markers of musculoskeletal ageing and osteoarthritis

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation

Introduction: Ribosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation. Materials: SnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling. Results: As a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5-6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation. Conclusion: The expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases

Plasma and liver lipid levels.

<p>(<i>A</i>) Plasma total triglycerides (TG), total cholesterol (TC) and free fatty acids (FFA) after 3 months on a chow or HFC diet in Wt-tp and caspase-1/11^-/--tp mice. (<i>B</i>) Liver TG, TC and FFA after 3 months on chow or HFC diet. (C) Representative images (100x magnification) of the Oil Red O staining of liver sections from Wt-tp and caspase-1/11^-/--tp mice on HFC for 3 months. Data are represented as mean +/- SEM.</p

Parameters for cholesterol efflux and autophagy.

<p>(<i>A</i>) Gene expression analysis of ABC-transporter a1 (Abca1) and ABC-transporter g1 (Abcg1) in isolated KCs. (<i>B</i>, <i>C</i>) Representative electron microscopy images of KCs from Wt-tp (<i>B</i>) and caspase-1/11^-/--tp (<i>C</i>) mice on HFC diet. Double membranes surrounding lipid droplets (autophagosomes) are indicated by arrows. (<i>D</i>) Average autophagosome number per Kupffer cell. (<i>E</i>) Gene expression analysis of lysosomal-associated membrane protein 2 (Lamp2) in isolated KCs. Data are represented as mean +/- SEM and were set relative to the Wt-tp group on chow diet. *Significantly different from Wt-tp on HFC diet. **p<0.01, ***p<0.001. </p

Foamy appearance of Kupffer cells, Cathepsin D activity and electron microscopy of KCs.

<p>(<i>A</i>, <i>B</i>) Representative pictures of CD68 staining (200x magnification) after 3 months of HFC feeding in Wt-tp and caspase-1/11^-/--tp mice, respectively. (<i>C</i>) Gene expression analysis of macrophage and Kupffer cell marker Cd68 in liver of Wt-tp and caspase-1/11^-/--tp mice. (<i>D</i>) Activity of the lysosomal enzyme cathepsin D in livers of Wt-tp and caspase-1/11^-/--tp mice. (<i>E</i>) Scoring for the amount of cholesterol crystallization inside Kupffer cells; 0 indicates that no cholesterol crystals were present while 5 indicates the highest amount of cholesterol crystals present. (<i>F</i>) Representative electron microscopy pictures of KCs from Wt-tp and caspase-1/11^-/--tp mice on HFC diet. Lysosomes are indicated in black by acid phosphatase staining and cholesterol crystals are indicated by arrows. Data are represented as mean +/- SEM and were set relative to the Wt-tp group on chow diet. *Significantly different from Wt-tp on HFC diet. *p<0.05.</p

Parameters of hepatic inflammation.

<p>(<i>A</i>-<i>C</i>) Liver sections were stained for infiltrated macrophages and neutrophils (Mac-1), neutrophils (NIMP) and T cells (CD3⁺). (<i>D</i>) Representative pictures of Mac-1 staining (200x magnification) after 3 months of HFC feeding in Wt-tp and caspase-1/11^-/--tp mice. (<i>E</i>) Gene expression of pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β), interleukin 18 (IL18) and chemokine (C-C motif) ligand 5 (CCL5) in isolated KCs. Data are represented as mean +/- SEM and were set relative to the Wt-tp group on chow diet. * and ** Significantly different from Wt-tp on HFC diet. *p<0.05, **p<0.01.</p

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

<p>Background & Aims: While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr(-/-) mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.</p><p>Methods: Ldlr(-/-) mice were transplanted (tp) with wild-type (Wt) or caspase-1/11(-/-) (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11(-/-) mice were incubated with oxLDL for 24h and autophagy was assessed.</p><p>Results: In line with our hypothesis, caspase-1/11(-/-)-tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11(-/-)-tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11(-/-) mice had normal autophagy activity.</p><p>Conclusion: Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.</p>