Career situation of first and presenting authorPost-doctoral fellow.IntroductionOsteocytes derive from bone-forming osteoblasts and are located deep inside the bone matrix. They are encysted in cavities (lacunae) and form dendritic extensions to develop a dense sentinel network inside the bone. Osteocytes are not passive cells. They modulate bone remodelling through regulation of both osteoclast and osteoblast activity. Immature osteocytes express the transmembrane glycoprotein podoplanin (PDPN/gp38)1 which is important for dendrite elongation and osteocyte function.2 Moreover it has been reported that PDPN expression is regulated by inflammatory cytokines including TNFα, IL-6, IL-22, TGF-β1, IFN-γ3 and is highly expressed in synovial tissues from RA patients.4 However the role of PDPN in osteocytes during inflammatory disease has not previously been investigated.ObjectivesIn this study we investigated the effect of osteocyte-specific deletion of PDPN in the KBxN serum transfer (ST) mouse model of rheumatoid arthritis.MethodsDmp1 Cre mice were crossed with PDPNflox/flox mice to generate osteocyte specific conditional knockout mice as well as appropriate PDPNflox/fox controls. KBxN ST arthritis was used to study the effect of PDPN deletion on mouse arthritis progression. PDPN expression was assessed by immunohistochemistry. Osteoclast numbers were calculated by TRAP staining. Loss of cartilage and pannus formation was evaluated by Safranin-O and H and E staining. Standard microCT and synchrotron microCT analysis was used to assess bone density parameters, osteocyte location and bone erosions.ResultsPDPN is expressed in osteocytes at sites of bone erosions in inflamed joints of KBxN ST mouse model. Osteocyte-specific PDPN deletion has no effect on trabecular and cortical bone density parameters in mouse tibiae under resting conditions, as has been previously reported.2 However, loss of PDPN on osteocytes leads to significantly more bone erosions in the resolving phase of the KBxN ST model compared to non-deleted controls. Deletion of osteocyte PDPN has no effect on pannus formation, cartilage loss and osteoclast numbers.ConclusionsOsteocyte-derived PDPN has been suggested as an important sensor for bone damage and this study demonstrates its bone-protective function during inflammatory arthritis.ReferencesZhang KQ, et al. Molecular and Cellular Biology 2006;26:4539–4552.Staines KA, et al. J Cell Physiol 2017;232:3006–3019.Honma M, Minami-Hori M, Takahashi H, Iizuka HP. J Dermatol Sci 2012;65:134–140.Del Rey MJ, et al. Plos One 2014;9:e99607.Disclosure of InterestNone declared.
Chronic inflammatory conditions such as Rheumatoid Arthritis are characterised by over expression of pro-inflammatory factors resulting in prolonged and destructive inflammation. Dysregulation of endogenous anti-inflammatory control mechanisms contribute to this aetiology. The mRNA destabilising protein Tristetraprolin (TTP) targets pro-inflammatory transcripts for destruction, limiting the production of key inflammatory factors. Expression and activity of TTP are controlled via phosphorylation of serines 52 and 178. MAPK p38-dependent phosphorylation of these residues stabilises and inactivates TTP, promoting expression of inflammatory mediators and driving the on-phase of an inflammatory response. As MAPK p38 activity declines, accumulated TTP is activated by PP2A mediated dephosphorylation, driving the off-phase of the inflammatory response. We investigated the role of the TTP phosphorylation switch in inflammatory arthritis. Immuno-staining of human RA synovial biopsy tissue revealed abnormally high expression of TTP protein compared to control tissues, with highest expression in synovial macrophages. We hypothesise that TTP protein accumulates in a phosphorylated, inactive form, contributing to sustained expression of inflammatory mediators. To assess the therapeutic potential of targeting TTP we generated TTPaa/aa mice in which the two key serine residues in endogenous TTP are substituted. These mice produced significantly lower amounts of pro-inflammatory factors after systemic LPS challenge, due to constitutive mRNA destabilising activity of the mutant form of TTP, yet were still able to generate a protective immune response to bacterial infection. Intriguingly TTPaa/aa mice were protected from K/BxN induced inflammatory arthrtitis with no adverse histological pathology or bone remodelling compared to WT mice. Heterozygote mice, in which 20% of endogenous TTP is mutant, also demonstrated significant protection from inflammatory arthritis. Therefore, therapeutically altering the balance of activation in the total TTP pool could lead to a significant anti-inflammatory effect. An experimental reagent that activates TTP decreased clinical score, joint inflammation and bone erosions in K/BxN induced arthritis. Taken together, these data suggest that targeting of the equilibrium between phosphorylated (inactive) and dephosphorylated (active) TTP may exert therapeutic effects in arthritis without compromising immune function.
BackgroundInflammation, local joint destruction and systemic bone loss are common complications in patients with rheumatoid arthritis (RA). We have identified that localised pre-receptor activation of glucocorticoids (GC) by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is increased within sites of inflammation and surrounding tissues, such as synovium and bone. Whilst this greatly increases local bioavailability of cortisol, which supports resolution of inflammation, in chronic disease, GCs drive may drive catabolic pathways that contribute to joint destruction and systemic bone loss.ObjectivesTo determine the contribution of 11β-HSD1 activated glucocorticoids to joint destruction and inflammatory bone loss, we crossed an 11β-HSD1 null mouse onto a transgenic murine model of chronic polyarthritis (TNF-Tg) to generate TNF-tg11bKO mice.MethodsClinical measures of joint inflammation, mobility and behaviour were collected between 4 and 9 weeks of age. Paw swelling was determined using calliper measurements. Histology was assessed in formalin fixed sections following staining with haematoxylin and eosin, safranin O or TRAP staining. Juxta articular and systemic bone losses were measured by micro-Ct. synovitis was determined by Image J analysis of histology sections.Results11b-HSD1 was completely knocked out within sites of inflammation in the TNF-tg11βKO mouse. At 9 weeks, both clinical and inflammation scores were markedly exacerbated in TNF-tg11bKO animals relative to TNF-tg counterparts (inflammation score; TNF-tg, 4.3±2.26 versus TNF-tg11βKO, 11.08±0.86; p<0.001). This was supported by marked increases in joint swelling and juxta articular bone loss from these animals (erosion scores, TNFtg, 5.2±0.61 versus TNF-tg11βKO, 9.0±0.66; p<0.005). Closer examination of joint destruction revealed that the pannus was larger and more extensive within subchondral bone, whilst evidence of cartilage degradation was significantly worse in the TNF-tg11bKO mouse (synovitis size, TNFtg, 26 763 (AU) ±3200 versus TNF-tg11βKO, 530276±3225; p<0.005). Systemic bone loss determined by bone volume to tissue volume (BV/TV), trabecular thickness (TT) and trabecular number (TN) was also greatly exacerbated within the TNF-tg11bKO mouse (TNF-tg, BV/TV 5.7±0.75, TT 73.5±6.4, TN 0.00077±0.00004 versus TNF-tg11βKO BV/TV 1.8±0.36, TT 7359.77±3.7, TN 0.0003±0.00005; p<0.001, p<0.005, p<0.001 respectively).ConclusionsThis study demonstrates that rather than contributing to catabolic pathways of tissue destruction, local GC activation by 11b-HSD1 is critical in mediating the suppression inflammation, joint destruction, synovitis and inflammatory bone loss in this murine model of chronic polyarthritis.AcknowledgementsWe would like to thank Professor George Kollias (Hellenic Pasteur Institute, Athens, Greece) for providing the hTNFtg mice. This research was supported by the Arthritis Research UK grants (Reference: 19859 and 20843).Disclosure of InterestNone declared
Background and objectivesDuring synovial inflammation, platelets and their microparticles escape from the vasculature to fuel the synovial membrane with pro-inflammatory factors leading to the activation of synovial fibroblasts (SF) that actively contribute to joint damage.1 Patients with rheumatoid arthritis (RA) show an up-regulation of surface protein Podoplanin (PDPN) on SF.2,3 Although the function of PDPN is still poorly understood, recent data suggest that PDPN ligation to its ligand CLEC-2 can modulate cellular responses. Within the RA synovium, platelets are considered the sole source of CLEC-2.3 Despite these observations, clear experimental approaches that explore the role of PDPN/CLEC-2 interactions in RA are lacking.Materials and methodsPDPN expression by freshly isolated mouse synoviocytes was measured by flow cytometry during joint inflammation and after resolution. Tamoxifen-inducible Clec1b deletion mice (TIC mice) were used to assess the disease severity in absence of CLEC-2. CLEC-2 deletion was confirmed on circulating platelets by flow cytometry. Arthritis was induced by anti-collagen antibodies and LPS injections. The disease severity was monitored by body weight, clinical scores, ankle and paw thicknesses. Bone erosion and bone remodelling were studied by MicroCT scans and 3D reconstructions.ResultsJoint inflammation triggers PDPN up-regulation on SF and an accumulation of PDPN+ leucocytes in the synovium. These high levels of PDPN expression disappear when inflammation resolved. In absence of CLEC-2, arthritis is more severe, bone erosion and bone remodelling are more pronounced.ConclusionsIn this work, we provide the first in vivo evidence that PDPN/CLEC-2 interactions act to restrain arthritis by showing that ablation of CLEC-2 expression leads to worse arthritis, bone erosion and bone remodelling. These observations suggest that platelets, known for promoting joint inflammation, also contribute to the suppression of arthritis in a CLEC-2 dependent manner. The mechanisms underlying this anti-inflammatory process are currently under investigation.References Boilard E, et al. Nat Rev Rheumatol. 2012;8:534–542 Ekwall AK, et al. Arthritis Res Ther. 2011;13:R40 Del Rey MJ, et al. PLoS One. 2014;9:e0099607
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